EP3619325B1 - Methods for trapping and barcoding discrete biological units in hydrogel - Google Patents
Methods for trapping and barcoding discrete biological units in hydrogel Download PDFInfo
- Publication number
- EP3619325B1 EP3619325B1 EP18733327.3A EP18733327A EP3619325B1 EP 3619325 B1 EP3619325 B1 EP 3619325B1 EP 18733327 A EP18733327 A EP 18733327A EP 3619325 B1 EP3619325 B1 EP 3619325B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- barcode
- unit
- biological
- units
- hydrogel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000017 hydrogel Substances 0.000 title claims description 225
- 238000000034 method Methods 0.000 title claims description 193
- 150000007523 nucleic acids Chemical class 0.000 claims description 256
- 102000039446 nucleic acids Human genes 0.000 claims description 136
- 108020004707 nucleic acids Proteins 0.000 claims description 136
- 108020004414 DNA Proteins 0.000 claims description 132
- 230000003321 amplification Effects 0.000 claims description 101
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 101
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 99
- 210000004027 cell Anatomy 0.000 claims description 80
- 239000011159 matrix material Substances 0.000 claims description 74
- 239000012634 fragment Substances 0.000 claims description 45
- 238000012163 sequencing technique Methods 0.000 claims description 31
- 210000001623 nucleosome Anatomy 0.000 claims description 28
- 108010047956 Nucleosomes Proteins 0.000 claims description 27
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 239000011324 bead Substances 0.000 claims description 22
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 230000000379 polymerizing effect Effects 0.000 claims description 18
- 230000003407 synthetizing effect Effects 0.000 claims description 18
- 150000001720 carbohydrates Chemical class 0.000 claims description 15
- 230000017105 transposition Effects 0.000 claims description 15
- 229940088594 vitamin Drugs 0.000 claims description 12
- 229930003231 vitamin Natural products 0.000 claims description 12
- 235000013343 vitamin Nutrition 0.000 claims description 12
- 239000011782 vitamin Substances 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 11
- 210000004940 nucleus Anatomy 0.000 claims description 11
- 239000005515 coenzyme Substances 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 10
- 102000054766 genetic haplotypes Human genes 0.000 claims description 9
- 239000003446 ligand Substances 0.000 claims description 9
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 9
- 125000001165 hydrophobic group Chemical group 0.000 claims description 8
- 230000001413 cellular effect Effects 0.000 claims description 6
- 210000000349 chromosome Anatomy 0.000 claims description 6
- 241000700605 Viruses Species 0.000 claims description 4
- 229920002521 macromolecule Polymers 0.000 claims description 4
- 210000003763 chloroplast Anatomy 0.000 claims description 3
- 210000001808 exosome Anatomy 0.000 claims description 3
- 210000003470 mitochondria Anatomy 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims 3
- 108091034117 Oligonucleotide Proteins 0.000 description 88
- 239000000243 solution Substances 0.000 description 48
- 238000003752 polymerase chain reaction Methods 0.000 description 42
- 125000003729 nucleotide group Chemical group 0.000 description 37
- 239000002773 nucleotide Substances 0.000 description 35
- -1 polydimethylsiloxane Polymers 0.000 description 35
- 239000003599 detergent Substances 0.000 description 34
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 23
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 23
- 239000003153 chemical reaction reagent Substances 0.000 description 22
- 239000000047 product Substances 0.000 description 22
- 239000007787 solid Substances 0.000 description 22
- 239000000126 substance Substances 0.000 description 21
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 108010067770 Endopeptidase K Proteins 0.000 description 19
- 230000000295 complement effect Effects 0.000 description 19
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 17
- 230000009089 cytolysis Effects 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 16
- 238000006116 polymerization reaction Methods 0.000 description 16
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 15
- 238000003556 assay Methods 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000004205 dimethyl polysiloxane Substances 0.000 description 15
- 239000000499 gel Substances 0.000 description 15
- 150000004676 glycans Chemical class 0.000 description 15
- 238000007481 next generation sequencing Methods 0.000 description 15
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 15
- 229920001282 polysaccharide Polymers 0.000 description 15
- 239000005017 polysaccharide Substances 0.000 description 15
- 102100034343 Integrase Human genes 0.000 description 14
- 235000014633 carbohydrates Nutrition 0.000 description 14
- 239000011248 coating agent Substances 0.000 description 14
- 238000000576 coating method Methods 0.000 description 14
- 238000009792 diffusion process Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 13
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 12
- 229940072056 alginate Drugs 0.000 description 12
- 229920000615 alginic acid Polymers 0.000 description 12
- 235000010443 alginic acid Nutrition 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 238000007792 addition Methods 0.000 description 11
- 238000006073 displacement reaction Methods 0.000 description 11
- 238000010839 reverse transcription Methods 0.000 description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 10
- 230000006037 cell lysis Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 238000010586 diagram Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 238000005755 formation reaction Methods 0.000 description 9
- 230000000670 limiting effect Effects 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 229910052770 Uranium Inorganic materials 0.000 description 8
- 238000004132 cross linking Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 150000002772 monosaccharides Chemical class 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 108010017826 DNA Polymerase I Proteins 0.000 description 7
- 102000004594 DNA Polymerase I Human genes 0.000 description 7
- 102000003960 Ligases Human genes 0.000 description 7
- 108090000364 Ligases Proteins 0.000 description 7
- 108010012306 Tn5 transposase Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000000137 annealing Methods 0.000 description 7
- 238000013467 fragmentation Methods 0.000 description 7
- 238000006062 fragmentation reaction Methods 0.000 description 7
- 239000011521 glass Substances 0.000 description 7
- 239000010931 gold Substances 0.000 description 7
- 238000002844 melting Methods 0.000 description 7
- 230000008018 melting Effects 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 230000037452 priming Effects 0.000 description 7
- 239000008279 sol Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 239000004971 Cross linker Substances 0.000 description 6
- 238000010804 cDNA synthesis Methods 0.000 description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 238000003205 genotyping method Methods 0.000 description 6
- 239000010439 graphite Substances 0.000 description 6
- 229910002804 graphite Inorganic materials 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 108010090804 Streptavidin Proteins 0.000 description 5
- 108010006785 Taq Polymerase Proteins 0.000 description 5
- 238000011049 filling Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 229920000136 polysorbate Polymers 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 125000006850 spacer group Chemical group 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108020004635 Complementary DNA Proteins 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 4
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 4
- 108091034057 RNA (poly(A)) Proteins 0.000 description 4
- 238000003559 RNA-seq method Methods 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 4
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 235000010980 cellulose Nutrition 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 150000002016 disaccharides Chemical class 0.000 description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 4
- 238000007306 functionalization reaction Methods 0.000 description 4
- 238000013412 genome amplification Methods 0.000 description 4
- 230000000415 inactivating effect Effects 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- PXQPEWDEAKTCGB-UHFFFAOYSA-N orotic acid Chemical compound OC(=O)C1=CC(=O)NC(=O)N1 PXQPEWDEAKTCGB-UHFFFAOYSA-N 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 4
- 239000010703 silicon Substances 0.000 description 4
- 229910052710 silicon Inorganic materials 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 239000004593 Epoxy Chemical class 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 229920002148 Gellan gum Polymers 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 108010020764 Transposases Proteins 0.000 description 3
- 102000008579 Transposases Human genes 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 150000001718 carbodiimides Chemical class 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 102000013165 exonuclease Human genes 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000007834 ligase chain reaction Methods 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 229960003512 nicotinic acid Drugs 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 238000005192 partition Methods 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 238000005498 polishing Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 3
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 2
- KUDUQBURMYMBIJ-UHFFFAOYSA-N 2-prop-2-enoyloxyethyl prop-2-enoate Chemical compound C=CC(=O)OCCOC(=O)C=C KUDUQBURMYMBIJ-UHFFFAOYSA-N 0.000 description 2
- OTXNTMVVOOBZCV-UHFFFAOYSA-N 2R-gamma-tocotrienol Natural products OC1=C(C)C(C)=C2OC(CCC=C(C)CCC=C(C)CCC=C(C)C)(C)CCC2=C1 OTXNTMVVOOBZCV-UHFFFAOYSA-N 0.000 description 2
- QIGJYVCQYDKYDW-UHFFFAOYSA-N 3-O-alpha-D-mannopyranosyl-D-mannopyranose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(CO)OC(O)C1O QIGJYVCQYDKYDW-UHFFFAOYSA-N 0.000 description 2
- FUPYROAFYPQUSH-UHFFFAOYSA-N 3-[[3-(4-heptylphenyl)-3-hydroxypropyl]-dimethylazaniumyl]propane-1-sulfonate Chemical compound CCCCCCCC1=CC=C(C(O)CC[N+](C)(C)CCCS([O-])(=O)=O)C=C1 FUPYROAFYPQUSH-UHFFFAOYSA-N 0.000 description 2
- GUQQBLRVXOUDTN-XOHPMCGNSA-N 3-[dimethyl-[3-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CC(O)CS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 GUQQBLRVXOUDTN-XOHPMCGNSA-N 0.000 description 2
- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- ZWIADYZPOWUWEW-XVFCMESISA-N CDP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-XVFCMESISA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 229920000049 Carbon (fiber) Polymers 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 2
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- HAEJPQIATWHALX-KQYNXXCUSA-N ITP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(N=CNC2=O)=C2N=C1 HAEJPQIATWHALX-KQYNXXCUSA-N 0.000 description 2
- ABSPRNADVQNDOU-UHFFFAOYSA-N Menaquinone 1 Natural products C1=CC=C2C(=O)C(CC=C(C)C)=C(C)C(=O)C2=C1 ABSPRNADVQNDOU-UHFFFAOYSA-N 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 2
- FFDGPVCHZBVARC-UHFFFAOYSA-N N,N-dimethylglycine Chemical compound CN(C)CC(O)=O FFDGPVCHZBVARC-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical compound CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- RVSTWRHIGKXTLG-UHFFFAOYSA-N Pangamic acid Natural products CC(C)N(C(C)C)C(N(C(C)C)C(C)C)C(=O)OCC(O)C(O)C(O)C(O)C(O)=O RVSTWRHIGKXTLG-UHFFFAOYSA-N 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 229920001214 Polysorbate 60 Polymers 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 229920000297 Rayon Polymers 0.000 description 2
- 229920002305 Schizophyllan Polymers 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 2
- 229930003761 Vitamin B9 Natural products 0.000 description 2
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 2
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 2
- 229920002310 Welan gum Polymers 0.000 description 2
- 229920006397 acrylic thermoplastic Polymers 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- ANVAOWXLWRTKGA-XHGAXZNDSA-N all-trans-alpha-carotene Chemical compound CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1C(C)=CCCC1(C)C ANVAOWXLWRTKGA-XHGAXZNDSA-N 0.000 description 2
- RZFHLOLGZPDCHJ-DLQZEEBKSA-N alpha-Tocotrienol Natural products Oc1c(C)c(C)c2O[C@@](CC/C=C(/CC/C=C(\CC/C=C(\C)/C)/C)\C)(C)CCc2c1C RZFHLOLGZPDCHJ-DLQZEEBKSA-N 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 238000004422 calculation algorithm Methods 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 239000004917 carbon fiber Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 229920002301 cellulose acetate Polymers 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 229940045110 chitosan Drugs 0.000 description 2
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 2
- 239000005516 coenzyme A Substances 0.000 description 2
- 229940093530 coenzyme a Drugs 0.000 description 2
- 229910052681 coesite Inorganic materials 0.000 description 2
- 239000005289 controlled pore glass Substances 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 229910052906 cristobalite Inorganic materials 0.000 description 2
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-N dCTP Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO[P@](O)(=O)O[P@](O)(=O)OP(O)(O)=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-N 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 2
- 229960002086 dextran Drugs 0.000 description 2
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 108700003601 dimethylglycine Proteins 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 229960002061 ergocalciferol Drugs 0.000 description 2
- CWAFVXWRGIEBPL-UHFFFAOYSA-N ethoxysilane Chemical compound CCO[SiH3] CWAFVXWRGIEBPL-UHFFFAOYSA-N 0.000 description 2
- SFNALCNOMXIBKG-UHFFFAOYSA-N ethylene glycol monododecyl ether Chemical compound CCCCCCCCCCCCOCCO SFNALCNOMXIBKG-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 2
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 2
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 2
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- 235000010492 gellan gum Nutrition 0.000 description 2
- 239000000216 gellan gum Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229920000591 gum Polymers 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- YOZNUFWCRFCGIH-BYFNXCQMSA-L hydroxocobalamin Chemical compound O[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O YOZNUFWCRFCGIH-BYFNXCQMSA-L 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229920000592 inorganic polymer Polymers 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 125000003010 ionic group Chemical group 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- YFVGRULMIQXYNE-UHFFFAOYSA-M lithium;dodecyl sulfate Chemical compound [Li+].CCCCCCCCCCCCOS([O-])(=O)=O YFVGRULMIQXYNE-UHFFFAOYSA-M 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910044991 metal oxide Inorganic materials 0.000 description 2
- 150000004706 metal oxides Chemical class 0.000 description 2
- 229910052752 metalloid Inorganic materials 0.000 description 2
- 150000002738 metalloids Chemical class 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- ARYZCSRUUPFYMY-UHFFFAOYSA-N methoxysilane Chemical compound CO[SiH3] ARYZCSRUUPFYMY-UHFFFAOYSA-N 0.000 description 2
- 239000010445 mica Substances 0.000 description 2
- 229910052618 mica group Inorganic materials 0.000 description 2
- UMWKZHPREXJQGR-UHFFFAOYSA-N n-methyl-n-(2,3,4,5,6-pentahydroxyhexyl)decanamide Chemical compound CCCCCCCCCC(=O)N(C)CC(O)C(O)C(O)C(O)CO UMWKZHPREXJQGR-UHFFFAOYSA-N 0.000 description 2
- SBWGZAXBCCNRTM-CTHBEMJXSA-N n-methyl-n-[(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl]octanamide Chemical compound CCCCCCCC(=O)N(C)C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO SBWGZAXBCCNRTM-CTHBEMJXSA-N 0.000 description 2
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 239000002135 nanosheet Substances 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 229920002113 octoxynol Polymers 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000013307 optical fiber Substances 0.000 description 2
- 229920000620 organic polymer Polymers 0.000 description 2
- 150000001282 organosilanes Chemical class 0.000 description 2
- 229960005010 orotic acid Drugs 0.000 description 2
- ZQTHOIGMSJMBLM-BUJSFMDZSA-N pangamic acid Chemical compound CN(C)CC(=O)OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O ZQTHOIGMSJMBLM-BUJSFMDZSA-N 0.000 description 2
- 108700024047 pangamic acid Proteins 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 235000019175 phylloquinone Nutrition 0.000 description 2
- 239000011772 phylloquinone Substances 0.000 description 2
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 2
- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 description 2
- 229960001898 phytomenadione Drugs 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 229920001993 poloxamer 188 Polymers 0.000 description 2
- 229920001992 poloxamer 407 Polymers 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 229920001748 polybutylene Polymers 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 229920000139 polyethylene terephthalate Polymers 0.000 description 2
- 239000005020 polyethylene terephthalate Substances 0.000 description 2
- 229920000193 polymethacrylate Polymers 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920006324 polyoxymethylene Polymers 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 229940068965 polysorbates Drugs 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 229920002635 polyurethane Polymers 0.000 description 2
- 239000004814 polyurethane Substances 0.000 description 2
- 230000001915 proofreading effect Effects 0.000 description 2
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 2
- NHZMQXZHNVQTQA-UHFFFAOYSA-N pyridoxamine Chemical compound CC1=NC=C(CO)C(CN)=C1O NHZMQXZHNVQTQA-UHFFFAOYSA-N 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 239000002964 rayon Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000005060 rubber Substances 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- UQMGAWUIVYDWBP-UHFFFAOYSA-N silyl acetate Chemical class CC(=O)O[SiH3] UQMGAWUIVYDWBP-UHFFFAOYSA-N 0.000 description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 2
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 229910052682 stishovite Inorganic materials 0.000 description 2
- PTISTKLWEJDJID-UHFFFAOYSA-N sulfanylidenemolybdenum Chemical class [Mo]=S PTISTKLWEJDJID-UHFFFAOYSA-N 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- ISXSCDLOGDJUNJ-UHFFFAOYSA-N tert-butyl prop-2-enoate Chemical compound CC(C)(C)OC(=O)C=C ISXSCDLOGDJUNJ-UHFFFAOYSA-N 0.000 description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 2
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 2
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 2
- 239000011678 thiamine pyrophosphate Substances 0.000 description 2
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- AOBORMOPSGHCAX-DGHZZKTQSA-N tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 2
- 229960000984 tocofersolan Drugs 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 229910052905 tridymite Inorganic materials 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000019159 vitamin B9 Nutrition 0.000 description 2
- 239000011727 vitamin B9 Substances 0.000 description 2
- 235000001892 vitamin D2 Nutrition 0.000 description 2
- 239000011653 vitamin D2 Substances 0.000 description 2
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 2
- 235000005282 vitamin D3 Nutrition 0.000 description 2
- 239000011647 vitamin D3 Substances 0.000 description 2
- 235000019143 vitamin K2 Nutrition 0.000 description 2
- 239000011728 vitamin K2 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- 229940021056 vitamin d3 Drugs 0.000 description 2
- 229920001285 xanthan gum Polymers 0.000 description 2
- WGVKWNUPNGFDFJ-DQCZWYHMSA-N β-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C WGVKWNUPNGFDFJ-DQCZWYHMSA-N 0.000 description 2
- GZIFEOYASATJEH-VHFRWLAGSA-N δ-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-VHFRWLAGSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OFHCOWSQAMBJIW-AOSUDXALSA-N (1r,3s,5e)-5-[(2e)-2-[(1r,3as,7ar)-7a-methyl-1-[(2r)-6-methylheptan-2-yl]-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1/C[C@@H](O)C[C@H](O)C1=C OFHCOWSQAMBJIW-AOSUDXALSA-N 0.000 description 1
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- WDQLRUYAYXDIFW-RWKIJVEZSA-N (2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-4-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 WDQLRUYAYXDIFW-RWKIJVEZSA-N 0.000 description 1
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 description 1
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 1
- OAJLVMGLJZXSGX-SLAFOUTOSA-L (2s,3s,4r,5r)-2-(6-aminopurin-9-yl)-5-methanidyloxolane-3,4-diol;cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7 Chemical compound [Co+3].O[C@H]1[C@@H](O)[C@@H]([CH2-])O[C@@H]1N1C2=NC=NC(N)=C2N=C1.[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O OAJLVMGLJZXSGX-SLAFOUTOSA-L 0.000 description 1
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 description 1
- ZNOVTXRBGFNYRX-STQMWFEESA-N (6S)-5-methyltetrahydrofolic acid Chemical compound C([C@@H]1N(C=2C(=O)N=C(N)NC=2NC1)C)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-STQMWFEESA-N 0.000 description 1
- FGYKUFVNYVMTAM-UHFFFAOYSA-N (R)-2,5,8-trimethyl-2-(4,8,12-trimethyl-trideca-3t,7t,11-trienyl)-chroman-6-ol Natural products OC1=CC(C)=C2OC(CCC=C(C)CCC=C(C)CCC=C(C)C)(C)CCC2=C1C FGYKUFVNYVMTAM-UHFFFAOYSA-N 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N (R)-alpha-Tocopherol Natural products OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
- XUCIJNAGGSZNQT-JHSLDZJXSA-N (R)-amygdalin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O[C@@H](C#N)C=2C=CC=CC=2)O1 XUCIJNAGGSZNQT-JHSLDZJXSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- FEBUJFMRSBAMES-UHFFFAOYSA-N 2-[(2-{[3,5-dihydroxy-2-(hydroxymethyl)-6-phosphanyloxan-4-yl]oxy}-3,5-dihydroxy-6-({[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}methyl)oxan-4-yl)oxy]-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl phosphinite Chemical compound OC1C(O)C(O)C(CO)OC1OCC1C(O)C(OC2C(C(OP)C(O)C(CO)O2)O)C(O)C(OC2C(C(CO)OC(P)C2O)O)O1 FEBUJFMRSBAMES-UHFFFAOYSA-N 0.000 description 1
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 description 1
- PITRRWWILGYENJ-UHFFFAOYSA-N 2-[2-[2-[2-[2-(4-nonylphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCO)C=C1 PITRRWWILGYENJ-UHFFFAOYSA-N 0.000 description 1
- VEBJYBIQIYFEFN-UHFFFAOYSA-N 2-[2-[2-[2-[2-(4-octylphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCO)C=C1 VEBJYBIQIYFEFN-UHFFFAOYSA-N 0.000 description 1
- WIHIUFRJMOAJFO-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nonylphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO)C=C1 WIHIUFRJMOAJFO-UHFFFAOYSA-N 0.000 description 1
- NLMKTBGFQGKQEV-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hexadecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO NLMKTBGFQGKQEV-UHFFFAOYSA-N 0.000 description 1
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 description 1
- LBCZOTMMGHGTPH-UHFFFAOYSA-N 2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCO)C=C1 LBCZOTMMGHGTPH-UHFFFAOYSA-N 0.000 description 1
- RXXPAEGIPXPLPB-UHFFFAOYSA-N 2-[2-[4-(7-methyloctyl)phenoxy]ethoxy]ethanol Chemical compound CC(C)CCCCCCC1=CC=C(OCCOCCO)C=C1 RXXPAEGIPXPLPB-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- MSFSPUZXLOGKHJ-PGYHGBPZSA-N 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose Chemical compound OC(=O)[C@@H](C)O[C@@H]1[C@@H](N)C(O)O[C@H](CO)[C@H]1O MSFSPUZXLOGKHJ-PGYHGBPZSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- VHSHLMUCYSAUQU-UHFFFAOYSA-N 2-hydroxypropyl methacrylate Chemical compound CC(O)COC(=O)C(C)=C VHSHLMUCYSAUQU-UHFFFAOYSA-N 0.000 description 1
- JWUBBDSIWDLEOM-UHFFFAOYSA-N 25-Hydroxycholecalciferol Natural products C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2C1=CC=C1CC(O)CCC1=C JWUBBDSIWDLEOM-UHFFFAOYSA-N 0.000 description 1
- 239000003872 25-hydroxy-cholecalciferol Substances 0.000 description 1
- ODADKLYLWWCHNB-UHFFFAOYSA-N 2R-delta-tocotrienol Natural products OC1=CC(C)=C2OC(CCC=C(C)CCC=C(C)CCC=C(C)C)(C)CCC2=C1 ODADKLYLWWCHNB-UHFFFAOYSA-N 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- GACDQMDRPRGCTN-KQYNXXCUSA-N 3'-phospho-5'-adenylyl sulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OS(O)(=O)=O)[C@@H](OP(O)(O)=O)[C@H]1O GACDQMDRPRGCTN-KQYNXXCUSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- CLCSYZQBLQDRQU-UHFFFAOYSA-N 3-[3-(hexadecanoylamino)propyl-dimethylazaniumyl]propane-1-sulfonate Chemical compound CCCCCCCCCCCCCCCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O CLCSYZQBLQDRQU-UHFFFAOYSA-N 0.000 description 1
- WKALLSVICJPZTM-UHFFFAOYSA-N 3-[decyl(dimethyl)azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O WKALLSVICJPZTM-UHFFFAOYSA-N 0.000 description 1
- DIROHOMJLWMERM-UHFFFAOYSA-N 3-[dimethyl(octadecyl)azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O DIROHOMJLWMERM-UHFFFAOYSA-N 0.000 description 1
- QZRAABPTWGFNIU-UHFFFAOYSA-N 3-[dimethyl(octyl)azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O QZRAABPTWGFNIU-UHFFFAOYSA-N 0.000 description 1
- UTSXERRKRAEDOV-UHFFFAOYSA-N 3-[dimethyl-[3-(tetradecanoylamino)propyl]azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCCCCCCCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O UTSXERRKRAEDOV-UHFFFAOYSA-N 0.000 description 1
- TUBRCQBRKJXJEA-UHFFFAOYSA-N 3-[hexadecyl(dimethyl)azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O TUBRCQBRKJXJEA-UHFFFAOYSA-N 0.000 description 1
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PZPXDAEZSA-N 4β-mannobiose Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-PZPXDAEZSA-N 0.000 description 1
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 description 1
- YBJHBAHKTGYVGT-OOZYFLPDSA-N 5-[(3as,4r,6ar)-2-oxohexahydro-1h-thieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-OOZYFLPDSA-N 0.000 description 1
- CERZMXAJYMMUDR-QBTAGHCHSA-N 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid Chemical compound N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO CERZMXAJYMMUDR-QBTAGHCHSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- PVXPPJIGRGXGCY-TZLCEDOOSA-N 6-O-alpha-D-glucopyranosyl-D-fructofuranose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)C(O)(CO)O1 PVXPPJIGRGXGCY-TZLCEDOOSA-N 0.000 description 1
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- RYWSYCQQUDFMAU-UHFFFAOYSA-N Acetomenaphthone Chemical compound C1=CC=C2C(OC(=O)C)=CC(C)=C(OC(C)=O)C2=C1 RYWSYCQQUDFMAU-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- WNCAVNGLACHSRZ-KAMYIIQDSA-N Allithiamine Chemical compound C=CCSSC(/CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N WNCAVNGLACHSRZ-KAMYIIQDSA-N 0.000 description 1
- WNCAVNGLACHSRZ-UHFFFAOYSA-N Allithiamine Natural products C=CCSSC(CCO)=C(C)N(C=O)CC1=CN=C(C)N=C1N WNCAVNGLACHSRZ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 108010006591 Apoenzymes Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- HRQKOYFGHJYEFS-UHFFFAOYSA-N Beta psi-carotene Chemical compound CC(C)=CCCC(C)=CC=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C HRQKOYFGHJYEFS-UHFFFAOYSA-N 0.000 description 1
- QYOVMAREBTZLBT-KTKRTIGZSA-N CCCCCCCC\C=C/CCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO QYOVMAREBTZLBT-KTKRTIGZSA-N 0.000 description 1
- 235000021318 Calcifediol Nutrition 0.000 description 1
- MBLYZRMZFUWLOZ-UHFFFAOYSA-N Calcitroic acid Natural products C1CCC2(C)C(C(CC(O)=O)C)CCC2C1=CC=C1CC(O)CC(O)C1=C MBLYZRMZFUWLOZ-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229920002558 Curdlan Polymers 0.000 description 1
- 239000001879 Curdlan Substances 0.000 description 1
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical class NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- YTBSYETUWUMLBZ-UHFFFAOYSA-N D-Erythrose Natural products OCC(O)C(O)C=O YTBSYETUWUMLBZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-CBPJZXOFSA-N D-Gulose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O WQZGKKKJIJFFOK-CBPJZXOFSA-N 0.000 description 1
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 1
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 description 1
- GZIFEOYASATJEH-UHFFFAOYSA-N D-delta tocopherol Natural products OC1=CC(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- YTBSYETUWUMLBZ-IUYQGCFVSA-N D-erythrose Chemical compound OC[C@@H](O)[C@@H](O)C=O YTBSYETUWUMLBZ-IUYQGCFVSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 1
- SNPLKNRPJHDVJA-ZETCQYMHSA-N D-panthenol Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCCO SNPLKNRPJHDVJA-ZETCQYMHSA-N 0.000 description 1
- 235000004866 D-panthenol Nutrition 0.000 description 1
- 239000011703 D-panthenol Substances 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical compound OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Natural products OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 description 1
- YTBSYETUWUMLBZ-QWWZWVQMSA-N D-threose Chemical compound OC[C@@H](O)[C@H](O)C=O YTBSYETUWUMLBZ-QWWZWVQMSA-N 0.000 description 1
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical compound OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010001132 DNA Polymerase beta Proteins 0.000 description 1
- 102000001996 DNA Polymerase beta Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 description 1
- SBJKKFFYIZUCET-UHFFFAOYSA-N Dehydroascorbic acid Natural products OCC(O)C1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-UHFFFAOYSA-N 0.000 description 1
- UCTLRSWJYQTBFZ-UHFFFAOYSA-N Dehydrocholesterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCCC(C)C)CCC33)C)C3=CC=C21 UCTLRSWJYQTBFZ-UHFFFAOYSA-N 0.000 description 1
- 229920000045 Dermatan sulfate Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920002085 Dialdehyde starch Chemical class 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 206010056474 Erythrosis Diseases 0.000 description 1
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 1
- 102100029075 Exonuclease 1 Human genes 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- MPJKWIXIYCLVCU-UHFFFAOYSA-N Folinic acid Natural products NC1=NC2=C(N(C=O)C(CNc3ccc(cc3)C(=O)NC(CCC(=O)O)CC(=O)O)CN2)C(=O)N1 MPJKWIXIYCLVCU-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 229920000926 Galactomannan Polymers 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229920002581 Glucomannan Polymers 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920000569 Gum karaya Polymers 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 102100039869 Histone H2B type F-S Human genes 0.000 description 1
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 1
- 101001035372 Homo sapiens Histone H2B type F-S Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 description 1
- 229920000288 Keratan sulfate Polymers 0.000 description 1
- 208000007976 Ketosis Diseases 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VSOAQEOCSA-N L-altropyranose Chemical compound OC[C@@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-VSOAQEOCSA-N 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- UBORTCNDUKBEOP-UHFFFAOYSA-N L-xanthosine Natural products OC1C(O)C(CO)OC1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920001543 Laminarin Polymers 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- COTIXRRJLCSLLS-PIJUOVFKSA-N Lipoyllysine Chemical compound OC(=O)[C@H](N)CCCCNC(=O)CCCCC1CCSS1 COTIXRRJLCSLLS-PIJUOVFKSA-N 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- BAQMYDQNMFBZNA-UHFFFAOYSA-N N-biotinyl-L-lysine Natural products N1C(=O)NC2C(CCCCC(=O)NCCCCC(N)C(O)=O)SCC21 BAQMYDQNMFBZNA-UHFFFAOYSA-N 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710149004 Nuclease P1 Proteins 0.000 description 1
- 101710149086 Nuclease S1 Proteins 0.000 description 1
- 102000003832 Nucleotidyltransferases Human genes 0.000 description 1
- 108090000119 Nucleotidyltransferases Proteins 0.000 description 1
- DKXNBNKWCZZMJT-UHFFFAOYSA-N O4-alpha-D-Mannopyranosyl-D-mannose Natural products O=CC(O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O DKXNBNKWCZZMJT-UHFFFAOYSA-N 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N O6-alpha-D-Galactopyranosyl-D-galactose Natural products OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- 101150102573 PCR1 gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- DJWYOLJPSHDSAL-UHFFFAOYSA-N Pantethine Natural products OCC(C)(C)C(O)C(=O)NCCC(=O)NCCSSCCNC(=O)CCNC(=O)C(O)C(C)(C)CO DJWYOLJPSHDSAL-UHFFFAOYSA-N 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 229920002507 Poloxamer 124 Polymers 0.000 description 1
- 229920002508 Poloxamer 181 Polymers 0.000 description 1
- 229920002509 Poloxamer 182 Polymers 0.000 description 1
- 229920002516 Poloxamer 331 Polymers 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001219 Polysorbate 40 Polymers 0.000 description 1
- 229920002642 Polysorbate 65 Polymers 0.000 description 1
- 229920002651 Polysorbate 85 Polymers 0.000 description 1
- YUGCAAVRZWBXEQ-UHFFFAOYSA-N Precholecalciferol Natural products C=1CCC2(C)C(C(C)CCCC(C)C)CCC2C=1C=CC1=C(C)CCC(O)C1 YUGCAAVRZWBXEQ-UHFFFAOYSA-N 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- 108010019653 Pwo polymerase Proteins 0.000 description 1
- 241000205160 Pyrococcus Species 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 238000001237 Raman spectrum Methods 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- OVVGHDNPYGTYIT-VHBGUFLRSA-N Robinobiose Natural products O(C[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](C)O1 OVVGHDNPYGTYIT-VHBGUFLRSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- UQZIYBXSHAGNOE-USOSMYMVSA-N Stachyose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O2)O1 UQZIYBXSHAGNOE-USOSMYMVSA-N 0.000 description 1
- 241000934878 Sterculia Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 241000589499 Thermus thermophilus Species 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 108010001244 Tli polymerase Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- SHGAZHPCJJPHSC-NWVFGJFESA-N Tretinoin Chemical compound OC(=O)/C=C(\C)/C=C/C=C(C)C=CC1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-NWVFGJFESA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004892 Triton X-102 Polymers 0.000 description 1
- 229920004929 Triton X-114 Polymers 0.000 description 1
- 229920004923 Triton X-15 Polymers 0.000 description 1
- 229920004893 Triton X-165 Polymers 0.000 description 1
- 229920004894 Triton X-305 Polymers 0.000 description 1
- 229920004895 Triton X-35 Polymers 0.000 description 1
- 229920004896 Triton X-405 Polymers 0.000 description 1
- 229920004897 Triton X-45 Polymers 0.000 description 1
- 229920004898 Triton X-705 Polymers 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 229930003776 Vitamin B4 Natural products 0.000 description 1
- 229930003571 Vitamin B5 Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- DIPPFEXMRDPFBK-UHFFFAOYSA-N Vitamin D4 Natural products C1CCC2(C)C(C(C)CCC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C DIPPFEXMRDPFBK-UHFFFAOYSA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- PFRQBZFETXBLTP-UHFFFAOYSA-N Vitamin K2 Natural products C1=CC=C2C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C(=O)C2=C1 PFRQBZFETXBLTP-UHFFFAOYSA-N 0.000 description 1
- UBORTCNDUKBEOP-HAVMAKPUSA-N Xanthosine Natural products O[C@@H]1[C@H](O)[C@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-HAVMAKPUSA-N 0.000 description 1
- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical compound O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 description 1
- UDMBCSSLTHHNCD-DGPXGRDGSA-N [(2r,5r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-DGPXGRDGSA-N 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- MYBUGVXNAHWTOL-QGOAFFKASA-N acefurtiamine Chemical compound C=1N=C(C)N=C(N)C=1CN(C=O)C(\C)=C(/CCOC(=O)COC(=O)C)SC(=O)C1=CC=CO1 MYBUGVXNAHWTOL-QGOAFFKASA-N 0.000 description 1
- 229950002075 acefurtiamine Drugs 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000012644 addition polymerization Methods 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 108010045649 agarase Proteins 0.000 description 1
- 150000001323 aldoses Chemical class 0.000 description 1
- 229960002535 alfacalcidol Drugs 0.000 description 1
- 125000005210 alkyl ammonium group Chemical group 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
- 238000007844 allele-specific PCR Methods 0.000 description 1
- 229940087168 alpha tocopherol Drugs 0.000 description 1
- 229940064063 alpha tocotrienol Drugs 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- SRBFZHDQGSBBOR-STGXQOJASA-N alpha-D-lyxopyranose Chemical compound O[C@@H]1CO[C@H](O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-STGXQOJASA-N 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- SHZGCJCMOBCMKK-SXUWKVJYSA-N alpha-L-fucose Chemical compound C[C@@H]1O[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-SXUWKVJYSA-N 0.000 description 1
- 239000011795 alpha-carotene Substances 0.000 description 1
- 235000003903 alpha-carotene Nutrition 0.000 description 1
- ANVAOWXLWRTKGA-HLLMEWEMSA-N alpha-carotene Natural products C(=C\C=C\C=C(/C=C/C=C(\C=C\C=1C(C)(C)CCCC=1C)/C)\C)(\C=C\C=C(/C=C/[C@H]1C(C)=CCCC1(C)C)\C)/C ANVAOWXLWRTKGA-HLLMEWEMSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- XSSWMWJCSXVEJC-UHFFFAOYSA-N amino propane-1-sulfonate Chemical class CCCS(=O)(=O)ON XSSWMWJCSXVEJC-UHFFFAOYSA-N 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000007846 asymmetric PCR Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- PCCNIENXBRUYFK-UHFFFAOYSA-O azanium;cerium(4+);pentanitrate Chemical compound [NH4+].[Ce+4].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O PCCNIENXBRUYFK-UHFFFAOYSA-O 0.000 description 1
- BTNNPSLJPBRMLZ-LGMDPLHJSA-N benfotiamine Chemical compound C=1C=CC=CC=1C(=O)SC(/CCOP(O)(O)=O)=C(/C)N(C=O)CC1=CN=C(C)N=C1N BTNNPSLJPBRMLZ-LGMDPLHJSA-N 0.000 description 1
- 229960002873 benfotiamine Drugs 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 229940066595 beta tocopherol Drugs 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- FGYKUFVNYVMTAM-YMCDKREISA-N beta-Tocotrienol Natural products Oc1c(C)c2c(c(C)c1)O[C@@](CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)(C)CC2 FGYKUFVNYVMTAM-YMCDKREISA-N 0.000 description 1
- 235000013734 beta-carotene Nutrition 0.000 description 1
- 239000011648 beta-carotene Substances 0.000 description 1
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- DLRVVLDZNNYCBX-ZZFZYMBESA-N beta-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- BAQMYDQNMFBZNA-MNXVOIDGSA-N biocytin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCC[C@H](N)C(O)=O)SC[C@@H]21 BAQMYDQNMFBZNA-MNXVOIDGSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- JWUBBDSIWDLEOM-DTOXIADCSA-N calcidiol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)CCC1=C JWUBBDSIWDLEOM-DTOXIADCSA-N 0.000 description 1
- 229960002882 calcipotriol Drugs 0.000 description 1
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 description 1
- 229960005084 calcitriol Drugs 0.000 description 1
- GMRQFYUYWCNGIN-NKMMMXOESA-N calcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-NKMMMXOESA-N 0.000 description 1
- 235000020964 calcitriol Nutrition 0.000 description 1
- 239000011612 calcitriol Substances 0.000 description 1
- MBLYZRMZFUWLOZ-FEUSBDLHSA-N calcitroic acid Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CC(O)=O)C)=C\C=C1\C[C@H](O)C[C@@H](O)C1=C MBLYZRMZFUWLOZ-FEUSBDLHSA-N 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229960004203 carnitine Drugs 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000007156 chain growth polymerization reaction Methods 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 235000006279 cobamamide Nutrition 0.000 description 1
- 239000011789 cobamamide Substances 0.000 description 1
- ZIHHMGTYZOSFRC-UWWAPWIJSA-M cobamamide Chemical compound C1(/[C@](C)(CCC(=O)NC[C@H](C)OP(O)(=O)OC2[C@H]([C@H](O[C@@H]2CO)N2C3=CC(C)=C(C)C=C3N=C2)O)[C@@H](CC(N)=O)[C@]2(N1[Co+]C[C@@H]1[C@H]([C@@H](O)[C@@H](O1)N1C3=NC=NC(N)=C3N=C1)O)[H])=C(C)\C([C@H](C/1(C)C)CCC(N)=O)=N\C\1=C/C([C@H]([C@@]\1(CC(N)=O)C)CCC(N)=O)=N/C/1=C(C)\C1=N[C@]2(C)[C@@](C)(CC(N)=O)[C@@H]1CCC(N)=O ZIHHMGTYZOSFRC-UWWAPWIJSA-M 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 235000019316 curdlan Nutrition 0.000 description 1
- 229940078035 curdlan Drugs 0.000 description 1
- 235000000639 cyanocobalamin Nutrition 0.000 description 1
- 239000011666 cyanocobalamin Substances 0.000 description 1
- 229960002104 cyanocobalamin Drugs 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 235000020960 dehydroascorbic acid Nutrition 0.000 description 1
- 239000011615 dehydroascorbic acid Substances 0.000 description 1
- 235000010389 delta-tocopherol Nutrition 0.000 description 1
- BTNBMQIHCRIGOU-UHFFFAOYSA-N delta-tocotrienol Natural products CC(=CCCC(=CCCC(=CCCOC1(C)CCc2cc(O)cc(C)c2O1)C)C)C BTNBMQIHCRIGOU-UHFFFAOYSA-N 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 1
- 229940051593 dermatan sulfate Drugs 0.000 description 1
- VGONTNSXDCQUGY-UHFFFAOYSA-N desoxyinosine Natural products C1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 VGONTNSXDCQUGY-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960003949 dexpanthenol Drugs 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 125000004386 diacrylate group Chemical group 0.000 description 1
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical class N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 description 1
- 229910000071 diazene Inorganic materials 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 description 1
- ILYCWAKSDCYMBB-OPCMSESCSA-N dihydrotachysterol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1/C[C@@H](O)CC[C@@H]1C ILYCWAKSDCYMBB-OPCMSESCSA-N 0.000 description 1
- 229960000465 dihydrotachysterol Drugs 0.000 description 1
- 229940120503 dihydroxyacetone Drugs 0.000 description 1
- 125000005442 diisocyanate group Chemical class 0.000 description 1
- ZLFRJHOBQVVTOJ-UHFFFAOYSA-N dimethyl hexanediimidate Chemical class COC(=N)CCCCC(=N)OC ZLFRJHOBQVVTOJ-UHFFFAOYSA-N 0.000 description 1
- BHATUINFZWUDIX-UHFFFAOYSA-O dimethyl-(3-sulfopropyl)-tetradecylazanium Chemical compound CCCCCCCCCCCCCC[N+](C)(C)CCCS(O)(=O)=O BHATUINFZWUDIX-UHFFFAOYSA-O 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000010894 electron beam technology Methods 0.000 description 1
- 238000005370 electroosmosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- FGYKUFVNYVMTAM-MUUNZHRXSA-N epsilon-Tocopherol Natural products OC1=CC(C)=C2O[C@@](CCC=C(C)CCC=C(C)CCC=C(C)C)(C)CCC2=C1C FGYKUFVNYVMTAM-MUUNZHRXSA-N 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- HQPMKSGTIOYHJT-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2-diol Chemical compound OCCO.CC(O)CO HQPMKSGTIOYHJT-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229940093476 ethylene glycol Drugs 0.000 description 1
- 108010092809 exonuclease Bal 31 Proteins 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940013640 flavin mononucleotide Drugs 0.000 description 1
- 239000011768 flavin mononucleotide Substances 0.000 description 1
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 1
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000008191 folinic acid Nutrition 0.000 description 1
- 239000011672 folinic acid Substances 0.000 description 1
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 description 1
- 150000002243 furanoses Chemical class 0.000 description 1
- JTLXCMOFVBXEKD-FOWTUZBSSA-N fursultiamine Chemical compound C1CCOC1CSSC(\CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N JTLXCMOFVBXEKD-FOWTUZBSSA-N 0.000 description 1
- 229950006836 fursultiamine Drugs 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229960003082 galactose Drugs 0.000 description 1
- OTXNTMVVOOBZCV-YMCDKREISA-N gamma-Tocotrienol Natural products Oc1c(C)c(C)c2O[C@@](CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)(C)CCc2c1 OTXNTMVVOOBZCV-YMCDKREISA-N 0.000 description 1
- 239000011663 gamma-carotene Substances 0.000 description 1
- 235000000633 gamma-carotene Nutrition 0.000 description 1
- HRQKOYFGHJYEFS-RZWPOVEWSA-N gamma-carotene Natural products C(=C\C=C\C(=C/C=C/C=C(\C=C\C=C(/C=C/C=1C(C)(C)CCCC=1C)\C)/C)\C)(\C=C\C=C(/CC/C=C(\C)/C)\C)/C HRQKOYFGHJYEFS-RZWPOVEWSA-N 0.000 description 1
- 235000010382 gamma-tocopherol Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000054767 gene variant Human genes 0.000 description 1
- DLRVVLDZNNYCBX-CQUJWQHSSA-N gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-CQUJWQHSSA-N 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 150000002386 heptoses Chemical class 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 238000007849 hot-start PCR Methods 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 235000004867 hydroxocobalamin Nutrition 0.000 description 1
- 239000011704 hydroxocobalamin Substances 0.000 description 1
- 229960001103 hydroxocobalamin Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 150000002454 idoses Chemical class 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 238000007851 intersequence-specific PCR Methods 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 235000010494 karaya gum Nutrition 0.000 description 1
- 239000000231 karaya gum Substances 0.000 description 1
- 229940039371 karaya gum Drugs 0.000 description 1
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 1
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 1
- 150000002584 ketoses Chemical class 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 description 1
- 229960000511 lactulose Drugs 0.000 description 1
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 description 1
- QIGJYVCQYDKYDW-LCOYTZNXSA-N laminarabiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1O QIGJYVCQYDKYDW-LCOYTZNXSA-N 0.000 description 1
- DBTMGCOVALSLOR-VPNXCSTESA-N laminarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1O[C@@H]1[C@@H](O)C(O[C@H]2[C@@H]([C@@H](CO)OC(O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-VPNXCSTESA-N 0.000 description 1
- IZWSFJTYBVKZNK-UHFFFAOYSA-N lauryl sulfobetaine Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O IZWSFJTYBVKZNK-UHFFFAOYSA-N 0.000 description 1
- 229960001691 leucovorin Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 229960003208 levomefolic acid Drugs 0.000 description 1
- 235000007635 levomefolic acid Nutrition 0.000 description 1
- 239000011578 levomefolic acid Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000007898 magnetic cell sorting Methods 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229940100434 menadiol Drugs 0.000 description 1
- ZJTLZYDQJHKRMQ-UHFFFAOYSA-N menadiol Chemical compound C1=CC=CC2=C(O)C(C)=CC(O)=C21 ZJTLZYDQJHKRMQ-UHFFFAOYSA-N 0.000 description 1
- 125000000695 menaquinone group Chemical group 0.000 description 1
- DKHGMERMDICWDU-GHDNBGIDSA-N menaquinone-4 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 DKHGMERMDICWDU-GHDNBGIDSA-N 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000007855 methylation-specific PCR Methods 0.000 description 1
- JEWJRMKHSMTXPP-BYFNXCQMSA-M methylcobalamin Chemical compound C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O JEWJRMKHSMTXPP-BYFNXCQMSA-M 0.000 description 1
- 235000007672 methylcobalamin Nutrition 0.000 description 1
- 239000011585 methylcobalamin Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000007856 miniprimer PCR Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 238000007838 multiplex ligation-dependent probe amplification Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- UDCIYVVYDCXLSX-SDNWHVSQSA-N n-[(4-amino-2-methylpyrimidin-5-yl)methyl]-n-[(e)-5-hydroxy-3-(propyldisulfanyl)pent-2-en-2-yl]formamide Chemical compound CCCSS\C(CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N UDCIYVVYDCXLSX-SDNWHVSQSA-N 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- GCRLIVCNZWDCDE-UHFFFAOYSA-N n-methyl-n-(2,3,4,5,6-pentahydroxyhexyl)nonanamide Chemical compound CCCCCCCCC(=O)N(C)CC(O)C(O)C(O)C(O)CO GCRLIVCNZWDCDE-UHFFFAOYSA-N 0.000 description 1
- GCRLIVCNZWDCDE-SJXGUFTOSA-N n-methyl-n-[(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl]nonanamide Chemical compound CCCCCCCCC(=O)N(C)C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO GCRLIVCNZWDCDE-SJXGUFTOSA-N 0.000 description 1
- 229940052665 nadh Drugs 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 210000000441 neoplastic stem cell Anatomy 0.000 description 1
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- QIGJYVCQYDKYDW-NSYYTRPSSA-N nigerose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1O QIGJYVCQYDKYDW-NSYYTRPSSA-N 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- VJTXQHYNRDGLON-LTGZKZEYSA-N octotiamine Chemical compound COC(=O)CCCCC(SC(C)=O)CCSS\C(CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N VJTXQHYNRDGLON-LTGZKZEYSA-N 0.000 description 1
- 229950011324 octotiamine Drugs 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical group [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
- 229940055705 pangamic acid Drugs 0.000 description 1
- 229960000903 pantethine Drugs 0.000 description 1
- DJWYOLJPSHDSAL-ROUUACIJSA-N pantethine Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSSCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)CO DJWYOLJPSHDSAL-ROUUACIJSA-N 0.000 description 1
- 235000008975 pantethine Nutrition 0.000 description 1
- 239000011581 pantethine Substances 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 229960000987 paricalcitol Drugs 0.000 description 1
- BPKAHTKRCLCHEA-UBFJEZKGSA-N paricalcitol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](\C=C\[C@H](C)C(C)(C)O)C)=C\C=C1C[C@@H](O)C[C@H](O)C1 BPKAHTKRCLCHEA-UBFJEZKGSA-N 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 150000003003 phosphines Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- ONJQDTZCDSESIW-UHFFFAOYSA-N polidocanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO ONJQDTZCDSESIW-UHFFFAOYSA-N 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 229940093448 poloxamer 124 Drugs 0.000 description 1
- 229940085692 poloxamer 181 Drugs 0.000 description 1
- 229940093426 poloxamer 182 Drugs 0.000 description 1
- 229940116406 poloxamer 184 Drugs 0.000 description 1
- 229940044519 poloxamer 188 Drugs 0.000 description 1
- 229940044476 poloxamer 407 Drugs 0.000 description 1
- 238000012643 polycondensation polymerization Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 1
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 1
- 235000010989 polyoxyethylene sorbitan monostearate Nutrition 0.000 description 1
- 239000001818 polyoxyethylene sorbitan monostearate Substances 0.000 description 1
- 235000010988 polyoxyethylene sorbitan tristearate Nutrition 0.000 description 1
- 239000001816 polyoxyethylene sorbitan tristearate Substances 0.000 description 1
- 229940068977 polysorbate 20 Drugs 0.000 description 1
- 229940101027 polysorbate 40 Drugs 0.000 description 1
- 229940113124 polysorbate 60 Drugs 0.000 description 1
- 229940099511 polysorbate 65 Drugs 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 229940113171 polysorbate 85 Drugs 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- YUGCAAVRZWBXEQ-WHTXLNIXSA-N previtamin D3 Chemical compound C=1([C@@H]2CC[C@@H]([C@]2(CCC=1)C)[C@H](C)CCCC(C)C)\C=C/C1=C(C)CC[C@H](O)C1 YUGCAAVRZWBXEQ-WHTXLNIXSA-N 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229950007142 prosultiamine Drugs 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 238000003906 pulsed field gel electrophoresis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003215 pyranoses Chemical class 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 229960001327 pyridoxal phosphate Drugs 0.000 description 1
- 235000008151 pyridoxamine Nutrition 0.000 description 1
- 239000011699 pyridoxamine Substances 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- SIXLXDIJGIWWFU-UHFFFAOYSA-N pyritinol Chemical compound OCC1=C(O)C(C)=NC=C1CSSCC1=CN=C(C)C(O)=C1CO SIXLXDIJGIWWFU-UHFFFAOYSA-N 0.000 description 1
- 229960004986 pyritinol Drugs 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 229960003471 retinol Drugs 0.000 description 1
- 235000020944 retinol Nutrition 0.000 description 1
- 239000011607 retinol Substances 0.000 description 1
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- OVVGHDNPYGTYIT-BNXXONSGSA-N rutinose Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 OVVGHDNPYGTYIT-BNXXONSGSA-N 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- OABYVIYXWMZFFJ-ZUHYDKSRSA-M sodium glycocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 OABYVIYXWMZFFJ-ZUHYDKSRSA-M 0.000 description 1
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 1
- 229940045946 sodium taurodeoxycholate Drugs 0.000 description 1
- YXHRQQJFKOHLAP-FVCKGWAHSA-M sodium;2-[[(4r)-4-[(3r,5r,8r,9s,10s,12s,13r,14s,17r)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 YXHRQQJFKOHLAP-FVCKGWAHSA-M 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- UQZIYBXSHAGNOE-XNSRJBNMSA-N stachyose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)O2)O)O1 UQZIYBXSHAGNOE-XNSRJBNMSA-N 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- CKHJPWQVLKHBIH-ZDSKVHJSSA-N sulbutiamine Chemical compound C=1N=C(C)N=C(N)C=1CN(C=O)C(/C)=C(/CCOC(=O)C(C)C)SS\C(CCOC(=O)C(C)C)=C(\C)N(C=O)CC1=CN=C(C)N=C1N CKHJPWQVLKHBIH-ZDSKVHJSSA-N 0.000 description 1
- 229960003211 sulbutiamine Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940066765 systemic antihistamines substituted ethylene diamines Drugs 0.000 description 1
- 229960004907 tacalcitol Drugs 0.000 description 1
- BJYLYJCXYAMOFT-RSFVBTMBSA-N tacalcitol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CC[C@@H](O)C(C)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C BJYLYJCXYAMOFT-RSFVBTMBSA-N 0.000 description 1
- KUCOHFSKRZZVRO-UHFFFAOYSA-N terephthalaldehyde Chemical compound O=CC1=CC=C(C=O)C=C1 KUCOHFSKRZZVRO-UHFFFAOYSA-N 0.000 description 1
- FBWNMEQMRUMQSO-UHFFFAOYSA-N tergitol NP-9 Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCOCCOCCOCCOCCO)C=C1 FBWNMEQMRUMQSO-UHFFFAOYSA-N 0.000 description 1
- 150000003538 tetroses Chemical class 0.000 description 1
- 238000001248 thermal gelation Methods 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 238000007862 touchdown PCR Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 150000003641 trioses Chemical class 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000008979 vitamin B4 Nutrition 0.000 description 1
- 239000011579 vitamin B4 Substances 0.000 description 1
- 235000009492 vitamin B5 Nutrition 0.000 description 1
- 239000011675 vitamin B5 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- DIPPFEXMRDPFBK-JPWDPSJFSA-N vitamin D4 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CC[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C DIPPFEXMRDPFBK-JPWDPSJFSA-N 0.000 description 1
- RMDJVOZETBHEAR-LQYWTLTGSA-N vitamin D5 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CC[C@@H](CC)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C RMDJVOZETBHEAR-LQYWTLTGSA-N 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- QAOHCFGKCWTBGC-UHFFFAOYSA-N wybutosine Natural products C1=NC=2C(=O)N3C(CCC(NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O QAOHCFGKCWTBGC-UHFFFAOYSA-N 0.000 description 1
- QAOHCFGKCWTBGC-QHOAOGIMSA-N wybutosine Chemical compound C1=NC=2C(=O)N3C(CC[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QAOHCFGKCWTBGC-QHOAOGIMSA-N 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- UBORTCNDUKBEOP-UUOKFMHZSA-N xanthosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UUOKFMHZSA-N 0.000 description 1
- RZFHLOLGZPDCHJ-XZXLULOTSA-N α-Tocotrienol Chemical compound OC1=C(C)C(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1C RZFHLOLGZPDCHJ-XZXLULOTSA-N 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 239000011730 α-tocotrienol Substances 0.000 description 1
- 235000019145 α-tocotrienol Nutrition 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
- 239000011590 β-tocopherol Substances 0.000 description 1
- 235000007680 β-tocopherol Nutrition 0.000 description 1
- 239000011723 β-tocotrienol Substances 0.000 description 1
- FGYKUFVNYVMTAM-WAZJVIJMSA-N β-tocotrienol Chemical compound OC1=CC(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1C FGYKUFVNYVMTAM-WAZJVIJMSA-N 0.000 description 1
- 235000019151 β-tocotrienol Nutrition 0.000 description 1
- 239000002478 γ-tocopherol Substances 0.000 description 1
- QUEDXNHFTDJVIY-DQCZWYHMSA-N γ-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 QUEDXNHFTDJVIY-DQCZWYHMSA-N 0.000 description 1
- 239000011722 γ-tocotrienol Substances 0.000 description 1
- OTXNTMVVOOBZCV-WAZJVIJMSA-N γ-tocotrienol Chemical compound OC1=C(C)C(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1 OTXNTMVVOOBZCV-WAZJVIJMSA-N 0.000 description 1
- 235000019150 γ-tocotrienol Nutrition 0.000 description 1
- 239000002446 δ-tocopherol Substances 0.000 description 1
- 239000011729 δ-tocotrienol Substances 0.000 description 1
- ODADKLYLWWCHNB-LDYBVBFYSA-N δ-tocotrienol Chemical compound OC1=CC(C)=C2O[C@@](CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1 ODADKLYLWWCHNB-LDYBVBFYSA-N 0.000 description 1
- 235000019144 δ-tocotrienol Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/125—Specific component of sample, medium or buffer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
- C12Q2535/122—Massive parallel sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2547/00—Reactions characterised by the features used to prevent contamination
- C12Q2547/10—Reactions characterised by the features used to prevent contamination the purpose being preventing contamination
- C12Q2547/107—Use of permeable barriers, e.g. waxes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/149—Particles, e.g. beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/159—Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/185—Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
Definitions
- the present invention relates to methods for trapping and barcoding discrete biological units in a hydrogel.
- the present invention relates to methods for discrete biological units' expression analysis.
- the methods of the present invention can further be used for single-cell transcriptome profiling, genotyping, phasing and/or haplotyping.
- sample preparation sample prep
- sequencing e.g., sequencing and 3) bioinformatics.
- Microfluidics has been exploited to improve the first of the three requirements, sample prep, specifically by enabling high throughput (HT) parallelization of reactions and efficiencies of scale.
- HT high throughput
- One application that has an acute need for HT microfluidic sample prep is single cell gene expression analysis by RNA sequencing (single cell RNAseq). The reason for this is that the number of cells to be analyzed can range from hundreds to thousands and each workflow starts by first isolating single cells in individual reaction chambers. Thus, the HT parallelization reaction capacity of any microfluidic platform needs to match these cell number requirements.
- the first microfluidic platform to be commercialized for single cell RNAseq analysis was based on PDMS (polydimethylsiloxane) chip technology. Available versions of the platform are able to process tens to hundreds of cells.
- Cells from a suspension are isolated in nanolitre (nL) volume PDMS chambers and then lysed by the application of a lysis reagent through the opening of valve and access to the appropriate lysis reagent inlet. Valve opening and selection of specific reagent inlets are done at each subsequent step to consecutively achieve reverse transcription of the mRNA, adaptor sequence addition to the cDNA and PCR. Amplicons from single cell products are then harvested from the chip and processed in bulk to finish sample prep from sequencing.
- PDMS polydimethylsiloxane
- Water in oil droplet emulsions are another form of microfluidics. Compared to PDMS based technology, droplets have the advantage of providing a significant increase in reaction numbers. Throughput is only limited by the emulsion volume and the numbers increase proportionally with decreasing droplet size.
- WO2016130704 and WO2017075265 have also disclosed methods based on hydrogel encapsulation of single cells. However, no barcoded particle that is able to form a complex with the cell was taught. Rather, barcoded particles were contacted with the cell after cell lysis.
- WO2015200541 discloses methods of analyzing nucleic acid of a plurality of cells, by forming partitions in a microfluidic device, each partition comprising a single barcoded particle and a single cell. Upon cell lysis, the capture sequence of the barcoded particle can hybridize to the nucleic acid of the single cell in each partition.
- droplets Although removing the throughput bottleneck of the PDMS chip technology, droplets have other significant drawbacks.
- droplets and their monodisperse formation are incompatible with detergent levels that are used to lyse difficult-to-lyse cells (such as plant cells, certain bacteria, in particular gram + bacteria, molds, spores, yeasts, mycobacteria, etc.), access nuclei and perform a number of critical molecular biology steps.
- Second, performing multi-step molecular biology reactions is extremely difficult in droplets.
- multi-step droplet workflows significantly increases the complexity and cost of the microfluidic setup.
- droplet platforms require high-grade oils, sophisticated chips whose features are difficult to manufacture at industrial scale, and instruments to accommodate and administer precise flow control through those chips. All three elements required for droplet platforms, namely oils, chips, and instruments, create a burden for manufacturing and tech support and, importantly, significantly increase the costs to the end user, thus limiting widespread droplet technology adoption.
- the current invention is designed to eliminate the drawbacks of the existing technologies. Indeed, the inventors have surprisingly developed a new method for single cell gene expression analysis, that does not require PDMS chips or droplets, while preserving the key benefit of droplet platforms in being able to process greater than thousands of cells. Based on the use of a hydrogel platform, this new technology also resolves the three key problems associated with droplet technologies. First, any detergent level is supported by the hydrogel platform, creating the possibility of lysing any cell or nuclei, as well as supporting key biochemistry and molecular biology reactions. Second, multistep reactions can be performed with ease since soluble reagents can easily access the reactor space through the hydrogel. Subsequent reactions are performed by simply exchanging the majority solution in contact with the hydrogel. Third, there is no need for expensive oils, chips and/or droplet generation instruments. For automation, an instrument may be used to manage the hydrogel reactor platform, but is not required.
- PDMS and droplet technologies and the improvements of the hydrogel reactor platform are not restricted to the single cell gene expression space. They apply to any application where the substrate has multiple primer binding sites, such as single cell genomes and long naked DNA molecules that are used as substrates in phasing and genome structure applications.
- the molecular biology reactions vary according to the identity of the substrate and the output requirements of the sample prep method.
- the foundational methods to trap and barcode biological units in hydrogel remains unchanged.
- the present invention relates to a method for trapping discrete biological units in a hydrogel, said method comprising the steps of:
- the present invention further relates to a method for analyzing gene expression in discrete biological units, said method comprising the steps of:
- the present invention further relates to a method for analyzing the genotype in discrete biological units, said method comprising the steps of:
- the present invention further relates to a method for analyzing the haplotype of discrete biological units, said method comprising the steps of:
- the present invention further relates to a method for analyzing the epigenome in discrete biological units, said method comprising the steps of:
- the biological units are immobilized on a support.
- the barcode units are immobilized on a support.
- the biological units are immobilized on a support in a hydrogel layer.
- the barcode units are immobilized on a support in a hydrogel layer.
- the unique barcode is present in multiple clonal copies on each barcode unit.
- the unique barcode comprises a nucleic acid sequence barcode.
- the unique barcode comprises a nucleic acid sequence primer.
- the nucleic acid sequence primer comprises random nucleic acid sequence primers. In one embodiment, the nucleic acid sequence primer comprises specific nucleic acid sequence primers.
- the at least a means involved with binding biological units comprises proteins, peptides and/or fragments thereof; antibodies and/or fragments thereof; nucleic acids; carbohydrates; vitamins and/or derivatives thereof; coenzymes and/or derivatives thereof; receptor ligands and/or derivatives thereof; and/or hydrophobic groups.
- each barcode unit consists of a bead.
- the step of barcoding is carried out in the hydrogel matrix by primer template annealing.
- the step of barcoding is carried out in the hydrogel matrix by primer-directed extension.
- the step of barcoding is carried out in the hydrogel matrix by ligation.
- discrete biological units comprise cells, groups of cells, viruses, nuclei, mitochondria, chloroplasts, biological macromolecules, exosomes, chromosomes, contiguity preserved transposition DNA fragments and/or nucleic acid fragments.
- cells or groups of cells comprise cells in in vitro culture, stem cells, tumor cells, tissue biopsy cells, blood cells and tissue section cells.
- a monosaccharide is a monomer, or simple sugar, having a single chain or a single ring structure.
- Monosaccharides can be further classified by their structure and the number of carbon atoms in the ring or chain, such as aldoses, ketoses, pyranoses, furanoses, trioses, tetroses, pentoses, hexoses, and heptoses, among others.
- monosaccharides include, but are not limited to, N-acetylglucosamine, allose, altrose, arabinose, deoxyribose, dihydroxyacetone, erythrose, fructose, fucose, ⁇ -L-fucopyranose, galactose, ⁇ -D-galactopyranose, galacturonic acid, glucose (dextrose), glucuronic acid, glyceraldehyde, gulose, idose, lyxose, mannose, ⁇ -D-mannopyranose, mannuronic acid, neuraminic acid, psicose, rhamnose, ribose, ribulose, sorbose, tagatose, threose, xylose, and xylulose.
- Disaccharides are formed from two monosaccharides joined by glycosidic bonds.
- Examples of disaccharides include, but are not limited to, cellobiose, gentiobiose, isomaltose, lactose, lactulose, laminaribiose, maltose, mannobiose, melibiose, nigerose, rutinose, sucrose, trehalose, and xylobiose.
- Polysaccharides are polymers formed from two or more monosaccharides joined by glycosidic bonds. Polysaccharides formed from 3-10 monosaccharides are often called oligosaccharides.
- polysaccharides include, but are not limited to, agarose, alginate, amylopectin, amylose, carageenan, cellulose, chitin, chitohexanose, chitosan, chondroitin sulfate, curdlan, dermatan sulfate, dextran, dextrin, emulsan, furcellaran, galactomannan, glucomannan, gellan gum, glucosamine, glycogen, glycosaminoglycan, guar gum, gum arabic, heparan sulfate, heparin, hyaluronic acid, deacylated hyaluronic acid, inulin, isomaltulose, karaya gum, keratan sulfate, laminaran, locust bean gum, muramic acid, pectic acid, pectin, pullulan, pustulan, rhamsan gum, schizophyll
- glycoconjugates are carbohydrates covalently bonded to other chemical species such as, for example, proteins and lipids.
- glycoconjugates include, but are not limited to, glycolipids, glycopeptides, glycoproteins, lipopolysaccharides, and peptidoglycans.
- coenzymes include, but are not limited to, nicotinamide adenine dinucleotide (NAD), NADH, nicotinamide adenine dinucleotide phosphate (NADP), NADPH, adenosine triphosphate (ATP), phosphoadenylyl sulfate (PAPS), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine triphosphate (GTP), inosine triphosphate (ITP), thiamine pyrophosphate (TPP), flavin mononucleotide (EMM), flavin adenine dinucleotide (FAD), coenzyme-A (CoA), biocytin, tetrahydrofolic acid, coenzyme B12, lipoyllysine, 1,1-cis-retinal and 1,2,5-dihydroxycholecalciferol.
- NAD nicotinamide adenine
- polysorbates examples include, but are not limited to, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65 (Tween 65), polysorbate 80 (Tween 80) and polysorbate 85 (Tween 85).
- octylphenol ethoxylates include, but are not limited to, Triton X-15, Triton X-35, Triton X-45, Triton X-100, Triton X-102, Triton X-114, Triton X-165 (70%), Triton X-305 (70%), Triton X-405 (70%) and Triton X-705 (70%).
- glucamines include, but are not limited to, N-octanoyl-N-methylglucamine (MEGA-8), N-nonanoyl-N-methylglucamine (MEGA-9) and N-decanoyl-N-methylglucamine (MEGA-10).
- Lubrol examples include, but are not limited to, Lubrol WX, Lubrol PX, Lubrol 12A9, Lubrol 17A10, Lubrol 17A17, Lubrol N13 and Lubrol G.
- Brij examples include, but are not limited to, Brij 35, Brij 58, Brij 93, Brij 97, Brij C2, Brij S2, Brij L4, Brij C10, Brij O10, Brij S10, Brij 020, Brij S20, Brij L23 and Brij S100.
- Nonidet examples include, but are not limited to, Nonidet P40.
- poloxamer examples include, but are not limited to, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 184, poloxamer 188 (Pluronic F68), poloxamer 331, poloxamer 407 (Pluronic F127)
- Genapol examples include, but are not limited to, Genapol X-080, Genapol X-100 and Genapol C-100.
- Igepal examples include, but are not limited to, Igepal CA-210, Igepal CA-520, Igepal CA-630, Igepal CA-720, Igepal CO-520, Igepal CO-630, Igepal CO-720, Igepal CO-890 and Igepal DM-970.
- Zwitterionic detergents refer to detergents which have ionic groups, but no net charge.
- zwitterionic detergents include, but are not limited to, amidosulfobetaines, alkylbetaines and ammonio propanesulfonates such as amidosulfobetaine-14, amidosulfobetaine-16, 3-[(3-cholamidopropyl)dimethylarmnonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), 3-(4-heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate (C7BzO), EMPIGEN ® BB, 3-(N,N-dimethyloctylammonio)propanesulfonate inner salt, 3-(decyldimethylammonio)propanesulfonate inner salt, 3-(dodecyldimethylammonio)propanesulfonate inner
- TdT terminal nucleotidyl transferase activity of the enzyme results in non-template-directed addition of nucleotides to the 3' end of the nascent cDNA strand.
- An exogenously added "template switch oligonucleotide” anneals to the C-tract by a poly(G) primer site.
- the reverse transcriptase then switches templates from the mRNA to the template switch oligonucleotide, adding an "adaptor sequence" or "adaptor” to the first strand cDNA (i.e. "adaptering”).
- the adaptor sequence shares homology with the PCR handle.
- the present invention relates to methods for trapping and barcoding discrete biological units in a hydrogel.
- a plurality of biological units is bound on a support.
- a plurality of barcode units is bound on a support.
- the method comprises contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes. In one embodiment, the method further comprises contacting the biological unit/barcode unit complexes with a hydrogel solution. In one embodiment, the method further comprises polymerizing the hydrogel solution to embed the biological unit/barcode unit complex in a hydrogel matrix. In one embodiment, the method further comprises barcoding the biological unit's nucleic acid within each biological unit/barcode unit complex in the hydrogel matrix.
- the biological units and barcode units unbind after hydrogel polymerization, i.e., the biological unit/barcode unit complexes' binding chemistry is degraded.
- Techniques to break down complexes are well-known to the skilled artisan.
- biochemistry and molecular biology assays can be performed on biological units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on discrete biological units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on barcode units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on discrete barcode units trapped in a hydrogel according to the present invention. In one embodiment, the hydrogel can be depolymerized to allow for certain biochemistry and molecular biology assays in solution and/or in bulk.
- biochemistry and molecular biology assays include, but are not limited to, cell lysis, PCR, reverse transcription, nucleic acid hydrolyzing, decapping ( i.e ., hydrolysis of a 5' cap structure), transcriptome profiling (or transcriptomics), genotyping (or genomics), epigenome profiling (or epigenomics), phasing, and haplotyping.
- Hydrogels can be classified into physical and chemical hydrogels based on their cross-linking mechanism.
- hydrogels are prepared from at least one natural polymer. In one embodiment, hydrogels are prepared from at least one synthetic polymer. In one embodiment, hydrogels are prepared from at least one natural/synthetic hybrid polymer. In one embodiment, hydrogels are prepared from at least one natural polymer and at least one synthetic polymer.
- the hydrogels used in the present invention are physical hydrogels.
- Physical hydrogel crosslinks include, but are not limited to, entangled chains, hydrogen bonding, hydrophobic interaction and crystallite formation.
- Physical hydrogel can be synthesized by ionic interaction, crystallization, stereocomplex formation, hydrophobized polysaccharides, protein interaction and hydrogen bond.
- physical hydrogels are permanent. In one embodiment, physical hydrogels are reversible. In one embodiment, the hydrogels used in the present invention are chemical hydrogels.
- Chemical hydrogels crosslinks include, but are not limited to, covalent bounds. Chemical hydrogels can be synthesized by chain growth polymerization, addition and condensation polymerization and gamma and electron beam polymerization.
- chemical hydrogels are formed by polymerization of end-functionalized macromers.
- chemical hydrogels are permanent. In one embodiment, chemical hydrogels are reversible. In one embodiment, hydrogels are polysaccharide hydrogels.
- Polysaccharides include, but are not limited to, alginate, agarose, ⁇ -carrageenan, -carrageenan, chitosan, dextran, heparin, gellan, native gellan gum, rhamsan, deacetylated rhamsan, S-657, welan.
- polymerized polysaccharide hydrogels are formed by covalent crosslinking, ionic crosslinking, chemical conjugation, esterification and/or polymerization.
- polysaccharide hydrogel is alginate and polymerized alginate is formed by ionic crosslinking in presence of a divalent cation, such as calcium.
- hydrogels are protein-based hydrogels.
- Proteins include, but are not limited to, collagen, fibrin, gelatin, laminin.
- polymerized protein-based hydrogels are formed by thermal gelation.
- protein-based hydrogels are crosslinked using a crosslinker.
- Protein-based hydrogels' crosslinkers include, but are not limited to, carbodiimide, cyanamide, dialdehyde starch, diimide, diisocyanate, dimethyl adipimidate, epoxy compounds, ethylaldehyde, formaldehyde, glutaraldehyde, glyceraldehyde, hexamethylenediamine, terephthalaldehyde and mixture thereof.
- hydrogels are polysaccharide hydrogels combined with proteins as described here above.
- hydrogels are nonbiodegradable synthetic hydrogels.
- Nonbiodegradable polymers include, but are not limited to, vinylated monomers and vinylated macromers, in particular, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, acrylamide, acrylic acid, N-isopropylacrylamide, poly N-isopropylacrylamide, methoxypolyethylene glycol monoacrylate.
- nonbiodegradable molecule polymerization requires at least one crosslinker.
- nonbiodegradable synthetic hydrogels are formed by copolymerization of a nonbiodegradable molecules and a crosslinker.
- Nonbiodegradable synthetic hydrogels' crosslinkers include, but are not limited to, N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate.
- nonbiodegradable molecule polymerization further requires at least one initiator, such as, e.g ., persulfate ions (ammonium persulfate, potassium persulfate and the like), ammonium cerium (IV) nitrate, tetramethylethylenediamine (TEMED).
- initiator such as, e.g ., persulfate ions (ammonium persulfate, potassium persulfate and the like), ammonium cerium (IV) nitrate, tetramethylethylenediamine (TEMED).
- the hydrogel can be depolymerized.
- depolymerization is meant a reaction during which the hydrogel returns in solution. As will clearly appear to the skilled person, this does not necessarily require extensive depolymerization and/or extensive breakage of crosslinks. The extent of depolymerization and/or breakage of crosslinks required to achieve gel-to-sol transition will depend on the nature of the hydrogel and can be readily determined by common methods.
- depolymerization of the hydrogel is chemical.
- depolymerization of the hydrogel is thermal.
- depolymerization of the hydrogel is enzymatic.
- depolymerization of the hydrogel can be achieved by divalent cation removal.
- hydrogels which can be depolymerized by divalent cation removal include, but are not limited to, alginate.
- depolymerization of the hydrogel can be achieved by addition of reducing agent.
- reducing agents include, but are not limited to, phosphines ( e.g ., tris(2-carboxyethyl)phosphine (TCEP)) and dithiothreitol (DTT).
- reducing agents include, but are not limited to, phosphines (e.g ., tris(2-carboxyethyl)phosphine (TCEP)) and dithiothreitol (DTT).
- hydrogels which can be depolymerized by addition of reducing agent include, but are not limited to, hydrogels copolymerized with a crosslinker such as nonbiodegradable synthetic hydrogels.
- depolymerization of the hydrogel can be achieved by thermal melting, i.e., melting upon increase of the temperature.
- the hydrogel used in the present invention is thermosensitive.
- thermosensitive is meant a hydrogel which, after being formed, depolymerizes if raised above the melting point of the at least one polymer, and reforms if cooled to room temperature or below its melting point.
- the hydrogel used in the present invention is thermoreversible.
- thermogel which, after being formed, depolymerizes if raised above the melting point of the at least one polymer and does not reform, even when cooled to room temperature or below its melting point.
- the melting point of the at least one polymer of the hydrogel is between about 20°C and about 200°C, preferably between about 25°C and about 100°C.
- the hydrogel has a pore size sufficiently small to trap a biological unit, a barcode unit and/or an analyte extracted or derived from a biological unit. In one embodiment, the hydrogel has a pore size sufficiently large to allow diffusion of biochemistry and molecular biology reagents.
- the hydrogel has a pore size ranging between about 1 nm and 1 ⁇ m, preferably between about 10 nm and 500 nm, more preferably between 25 nm and 250 nm.
- the hydrogel matrix is accessible to biochemistry and molecular biology reagents. In one embodiment, the hydrogel matrix has at least one surface accessible to biochemistry and molecular biology reagents. In one embodiment, the at least one surface accessible to biochemistry and molecular biology reagents is naturally occurring. In one embodiment, the at least one surface accessible to biochemistry and molecular biology reagents is shaped before, during and/or after hydrogel polymerization.
- composition, shape, form, and modifications of the barcode unit can be selected from a range of options depending on the application.
- Exemplary materials that can be used as a barcode unit in the present invention include, but are not limited to, acrylics, carbon (e.g ., graphite, carbon-fiber), cellulose (e.g ., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g ., agarose, SEPHAROSE TM or alginate), gels, glass ( e.g ., modified or functionalized glass), gold ( e.g ., atomically smooth Au(111)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides ( e.g ., SiO 2 , TiO 2 , stainless steel), metalloids, metals ( e.g ., atomically smooth Au(111)), mica, molybdenum sulfides, nanomaterials ( e.g ., highly oriented pyrolitic graphite (HOPG) nanosheets), nitrocellulose, NYLON TM , optical fibers
- the barcode unit is composed of a single material. In another embodiment, the barcode unit is composed of a mixture of several different materials.
- the barcode units used in the present invention can be simple square grids, checkerboard grids, hexagonal arrays and the like. Suitable barcode units also include, but are not limited to, beads, slides, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, microtiter plates such as 768-well, 384-well, 96-well, 48-well, 24-well, 12-well, 8-well, 6-well, 4-well, 1-well and the like. In various embodiments, the barcode unit may be biological, non-biological, organic, inorganic, or any combination thereof.
- a single barcode unit in a plurality of barcode units may be a minimal, indivisible part of said plurality of barcode units.
- a single barcode unit in a plurality of barcode units may be, e.g., a single square on a grid, a single bead in a population of beads, a single well in a microtiter plate, etc.
- a single barcode unit in a plurality of barcode units may be a minimal part of said plurality of barcode units, wherein a single binding event between a biological unit and a barcode unit occurs at the molecular level.
- a single barcode unit in a plurality of barcode units may be a part of said plurality of barcode units ranging from about 1 ⁇ m 2 to about 1 mm 2 , preferably from about 1 ⁇ m 2 to about 100 ⁇ m 2 , more preferably from about 1 ⁇ m 2 to about 50 ⁇ m 2 .
- this size range is chosen for manufacturability. In one embodiment, this size range is chosen to ensure the formation of biological unit/barcode unit complexes with a 1:1 ratio.
- the surface of the barcode unit can be modified according to methods known to the skilled artisan, to promote trapping or immobilization of biological units thereon.
- the barcode unit comprises reactive groups on its surface, such as carboxyl, amino, hydroxyl, epoxy, and the like.
- the barcode unit can have functional modifications, such as functional groups attached to its surface.
- the barcode unit used in the present invention is barcoded.
- each single barcode unit in a plurality of barcode units comprises a unique barcode. In one embodiment, each single barcode unit in a plurality of barcode units comprises clonal copies of a unique barcode.
- the barcode unit comprises at least one means involved with binding at least one biological unit.
- the barcode unit is a bead.
- the implementation of methods according to the present invention may rely on the downstream identification of each discrete biological unit and/or of the reactional analytes bound to each barcode unit. Therefore, it may be desirable to add at least one identifier or barcode to the barcode unit, in order to convey information about the source or origin of the biological unit and/or of an analyte within a sample, such as for example, a nucleic acid sequence extracted or derived from a discrete biological unit.
- the barcode unit is barcoded. In one embodiment, each single barcode unit in a plurality of barcode units comprises a unique barcode. In one embodiment, each single barcode unit in a plurality of barcode units comprises clonal copies of a unique barcode.
- Barcodes may be of a variety of different formats, including labels, tags, probes, and the like.
- the barcode unit is optically barcoded. In one embodiment, the barcode unit is non-optically barcoded. In one embodiment, the barcode unit is optically and non-optically barcoded.
- Optical barcodes include, but are not limited to, chromophores, fluorophores, quantum dots, styrene monomers, and combination thereof, which can be identified, e.g., by their spectrum such as Raman spectrum or electromagnetic spectrum; and/or by their intensity of color.
- Non-optical barcodes include, but are not limited to, biomolecular sequences such as DNA, RNA and/or protein sequences, which can be identified, e.g., by sequencing.
- the number of unique barcodes used in the present invention ranges from about 2 to about 10 12 .
- the number of clonal copies of each unique barcode comprised in each single barcode unit in a plurality of barcode units ranges from about 2 to about 10 12 .
- the barcode unit according to the present invention comprises non-optical barcodes. In one embodiment, the barcode unit according to the present invention comprises nucleic acid barcodes. In one embodiment, the nucleic acid barcode is single stranded. In one embodiment, the nucleic acid barcode is double stranded. In one embodiment, the nucleic acid barcode is single and/or double stranded. In one embodiment, the barcode unit according to the present invention comprises DNA barcodes. In one embodiment, the barcode unit according to the present invention comprises RNA barcodes. In one embodiment, the barcode unit according to the present invention comprises a mixture of DNA and RNA barcodes.
- the nucleic acid barcode according to the present invention comprises from 5 to 20 nucleotides, preferably from 8 to 16 nucleotides.
- the barcode unit comprises a plurality of unique nucleic acid sequences, i.e., clonal copies of a unique barcode.
- said unique nucleic acid sequences are degenerate sequences. In one embodiment, said unique nucleic acid sequences are based on combinatorial chemistry.
- barcodes on a support are well known to the skilled artisan, and include without limitation, replication of bound primers in a combinatorial fashion, ligation of adaptors in a combinatorial fashion, and chemical addition of nucleotides in a combinatorial fashion.
- said unique nucleic acid sequences are amplified on the barcode unit such that each single barcode unit in a plurality of barcode units is coated with clonal copies of a starting nucleic acid sequence.
- the covalent attachment of nucleic acid barcodes to the barcode unit is carried out directly during synthesis of the barcodes. In one embodiment, the covalent attachment of nucleic acid barcodes to the barcode unit is carried out after synthesis of the barcode.
- barcoding of the biological unit's nucleic acid is achieved by primer template annealing of the barcode to the biological unit's nucleic acid. In one embodiment, barcoding of the biological unit's nucleic acid is achieved by primer-directed extension of the barcode to the biological unit's nucleic acid. In one embodiment, barcoding of the biological unit's nucleic acid is achieved by ligation of the barcode to the biological unit's nucleic acid.
- the implementation of the methods according to the present invention may rely on the immobilization, replication, extension and/or amplification of nucleic acid sequences of or from the biological units. Therefore, it may be desirable to add at least one nucleic acid sequence primer to the barcode unit, preferably at least one nucleic acid sequence primer to each single barcode unit in a plurality of barcode units, in order to immobilize, replicate, extend and/or amplify genetic information of or from the biological units.
- the nucleic acid sequence primer is single-stranded. In one embodiment, the nucleic acid sequence primer is double-stranded. In one embodiment, the nucleic acid sequence primer is single-stranded and/or double-stranded.
- the nucleic acid sequence primer is a degenerate (i.e., random) nucleic acid sequence primer. In one embodiment, the nucleic acid sequence primer is specific to a nucleic acid sequence of interest. In one embodiment, the nucleic acid sequence primer can prime at multiple locations of the nucleic acid sequences of or from the biological units. In one embodiment, the nucleic acid sequences of or from the biological units comprise multiple priming sites.
- the nucleic acid sequence primer comprises a poly-dT sequence. In one embodiment, the nucleic acid sequence primer comprises a poly-dU sequence. Accordingly, the nucleic acid sequence primer is specific to a poly-A sequence. Poly-A sequences may be found, e.g., on the 3' end of mRNAs, within the poly-A tail.
- the nucleic acid sequence primer comprises the sequence (dT) n VN, wherein n ranges from 5 to 50, V represents any nucleotide but T/U ( i.e ., A, C or G), and N represents any nucleotide ( i.e ., A, T/U, C or G).
- the nucleic acid sequence primer comprises the sequence (dU) n VN, wherein n ranges from 5 to 50, V represents any nucleotide but T/U ( i.e ., A, C or G), and N represents any nucleotide ( i.e ., A, T/U, C or G).
- the nucleic acid sequence primer is specific to a (A) n BN sequence, wherein n ranges from 5 to 50, B represents any nucleotide but A ( i.e ., T/U, C or G), and N represents any nucleotide ( i.e ., A, T/U, C or G).
- (A) n BN sequences may be found, e.g., on the 3' end of mRNAs, overlapping between the poly-A tail and the 3' UTR or CDS.
- the nucleic acid sequence primer comprises a poly-I sequence. Accordingly, the nucleic acid sequence primer is non-specific and can prime to any nucleic acid sequence of or from the biological units. In one embodiment, the nucleic acid sequence primer comprises from 5 to 50 nucleotides, preferably from 5 to 30 nucleotides.
- the covalent attachment of nucleic acid sequence primers to the barcode unit is carried out directly during synthesis of the nucleic acid sequence primers. In one embodiment, the covalent attachment of nucleic acid sequence primers to the barcode unit is carried out after synthesis of the nucleic acid sequence primers.
- the barcode unit comprises at least one oligonucleotide.
- the at least one oligonucleotide is a DNA oligonucleotide. In one embodiment, the at least one oligonucleotide is a RNA oligonucleotide. In one embodiment, the at least one oligonucleotide is a DNA/RNA hybrid oligonucleotide.
- the at least one oligonucleotide is single-stranded. In one embodiment, the at least one oligonucleotide is double-stranded. In one embodiment, the at least one oligonucleotide is single-stranded and/or double-stranded.
- the at least one oligonucleotide comprises at least one nucleic acid barcode and at least one nucleic acid sequence primer. In one embodiment, the at least one oligonucleotide comprises from 5' to 3' at least one nucleic acid barcode and at least one nucleic acid sequence primer. In one embodiment, the at least one oligonucleotide comprises from 5' to 3' at least one nucleic acid sequence primer and at least one nucleic acid barcode. In one embodiment, the nucleic acid barcodes are identical across all oligonucleotides on the surface of a given barcode unit.
- the nucleic acid barcodes are different across oligonucleotides on the surface of one barcode unit with respect to another barcode unit.
- the nucleic acid sequence primer is identical across all oligonucleotides on the surface of a given barcode unit.
- the nucleic acid sequence primer is different across all oligonucleotides on the surface of a given barcode unit.
- the nucleic acid sequence primer is identical across all oligonucleotides and barcode units.
- the nucleic acid barcode comprises from 5 to 20 nucleotides, preferably from 8 to 16 nucleotides.
- the nucleic acid sequence primer comprises from 5 to 50 nucleotides, preferably from 5 to 30 nucleotides.
- the at least one oligonucleotide further comprises a PCR handle sequence.
- the PCR handle sequence is identical across all oligonucleotides and barcodes units.
- the PCR handle sequence comprises from 10 to 30 nucleotides, preferably from 15 to 25 nucleotides.
- the at least one oligonucleotide further comprises a unique molecular identifier sequence.
- the unique molecular identifier sequence is different across all oligonucleotides on the surface of a given barcode unit.
- the unique molecular identifier sequence comprises from 10 to 30 nucleotides, preferably from 15 to 25 nucleotides.
- the at least one oligonucleotide further comprises a spacer region.
- the at least one oligonucleotide comprises, from 5' to 3' ( i.e . , from proximal to distal with regard to the surface of the barcode unit):
- the at least one oligonucleotide comprises, from 3' to 5' ( i.e ., from distal to proximal with regard to the surface of the barcode unit):
- the covalent attachment of nucleic acid oligonucleotides to the barcode unit is carried out directly during synthesis of the nucleic acid oligonucleotides. In one embodiment, the covalent attachment of nucleic acid oligonucleotides to the barcode unit is carried out after synthesis of the nucleic acid oligonucleotides.
- the implementation of the methods according to the present invention may rely on the binding and/or the immobilization of a biological unit on the barcode unit. Therefore, it may be desirable to add at least one means for binding a biological unit to the barcode unit, in order to trap discrete biological units.
- the binding and/or the immobilization of a biological unit to the barcode unit is aspecific. In one embodiment, the binding and/or the immobilization of a biological unit to the barcode unit is specific. In one embodiment, the binding and/or the immobilization of a biological unit on the barcode unit requires the presence of at least one means for binding a barcode unit on the biological unit.
- Means for binding a biological unit and/or means for binding a barcode unit comprise, but is not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, a hydrophobic group.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a protein and/or at least a peptide.
- proteins or peptides include, but are not limited to, antibodies (e.g., IgA, IgD, IgE, IgG, and IgM) and fragments thereof, including, but not limited to, Fab fragments, F(ab') 2 fragments, scFv fragments, diabodies, triabodies, scFv-Fc fragments, minibodies; protein A, protein G, avidin, streptavidin, receptors and fragments thereof, and ligands and fragments thereof.
- antibodies e.g., IgA, IgD, IgE, IgG, and IgM
- fragments thereof including, but not limited to, Fab fragments, F(ab') 2 fragments, scFv fragments, diabodies, triabodies, scFv-Fc fragments, mini
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a nucleic acid.
- nucleic acids include, but are not limited to, DNA, RNA and artificial nucleic acids, such as nucleic acids comprising inosine, xanthosine, wybutosine, and/or analogs thereof.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a carbohydrate.
- carbohydrates include, but are not limited to, monosaccharides, disaccharides and polysaccharides.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a vitamin.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a coenzyme.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a receptor ligand.
- the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a hydrophobic group.
- hydrophobic groups include, but are not limited to, alkyl groups having from about 2 to about 8 carbon atoms, such as an ethyl, propyl, butyl, pentyl, heptyl, or octyl and isomeric forms thereof; or aryl groups such as phenyl, benzyl or naphthyl.
- the coating may be an all-over coating, i.e., completely covering the barcode unit, or may be a partial coating, i . e., covering only parts of the barcode unit.
- coating of a barcode unit with a means for binding a biological unit requires functionalization of the barcode unit.
- functionalized barcode units include, but are not limited to, amino-functionalized barcode units, carboxyl-functionalized barcode units, hydroxyl-functionalized barcode units and epoxy-functionalized barcode units.
- Techniques to functionalize a barcode unit are well-known in the art and include, but are not limited to, organosilane crosslinking, such as methoxysilane, ethoxysilane and acetoxysilane derivatives.
- Examples of techniques for coating a barcode unit with a means for binding a biological unit include, but are not limited to, adsorption and covalent attachment. Covalent attachment may be performed on functionalized barcode units, using coupling agents such as carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfo-NHS, dimethylaminopropyl (DEAP), glutaraldehyde, aldehyde, sodium cyanoborohydride (NaCNBH 3 ), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), dithiothreitol (DTT), and/or cyanogen bromide (BrNC).
- EDC carbodiimide
- NHS N-hydroxysuccinimide
- sulfo-NHS dimethylaminopropyl
- DEP dimethylaminopropyl
- SPDP succinimidyl 3-(2-pyridyldithio)propionate
- the biological unit is incubated with at least one antibody prior to the binding and/or the immobilization on the barcode unit.
- the at least one antibody is specific towards the biological unit.
- the at least one antibody is functionalized. Examples of functionalization include, but are not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, and a hydrophobic group.
- the antibody is biotinylated, i .
- the implementation of the methods according to the present invention may rely on the binding and/or the immobilization of a single biological unit on a single barcode unit. Therefore, it may be desirable to prevent more than one biological unit from binding to each barcode unit; or alternatively, to prevent more than one barcode unit from binding to each biological unit.
- the methods according to the present invention provides means for ensuring, selecting and/or purifying biological unit/barcode unit complexes with a 1:1 ratio.
- the methods according to the present invention also provides means for forming biological unit/barcode unit complexes with a 1:1 ratio.
- the methods of the present invention comprise a step of selection and/or purification of biological unit/barcode unit complexes with a 1:1 ratio.
- a plurality of biological units may be contacted with a plurality of barcode units to form biological unit/barcode unit complexes, which may be further selected and/or purified.
- the methods of the present invention comprise a means for forming biological unit/barcode unit complexes with a 1:1 ratio.
- the biological units are bound to a support.
- the barcode units are bound to a support.
- Binding a plurality of biological units to a support prior to contacting them with a plurality of barcode units creates hindrance and allows the support to act as an impediment, preventing multiple binding of barcode units to a single biological unit. It may thus be desirable to use larger barcode units with respect to the biological units. Additionally, a limiting concentration of barcode units with respect to the biological units may be used to ensure the binding of at most one barcode unit per biological unit.
- binding a plurality of barcode units to a support prior to contacting them with a plurality of biological units creates hindrance and allows the support to act as an impediment, preventing multiple binding of biological units to a single barcode unit. It may thus be desirable to use smaller barcode units with respect to the biological units. Additionally, a limiting concentration of biological units with respect to the barcode units may be used to ensure the binding of at most one biological unit per barcode unit.
- composition, shape, form, and modifications of the support can be selected from a range of options depending on the application.
- Exemplary materials that can be used as a support in the present invention include, but are not limited to, acrylics, carbon (e.g ., graphite, carbon-fiber), cellulose (e.g ., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g ., agarose, SEPHAROSE TM or alginate), gels, glass ( e.g ., modified or functionalized glass), gold ( e.g ., atomically smooth Au(111)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides ( e.g ., SiO 2, TiO 2 , stainless steel), metalloids, metals ( e.g ., atomically smooth Au(111)), mica, molybdenum sulfides, nanomaterials ( e.g ., highly oriented pyrolitic graphite (HOPG) nanosheets), nitrocellulose, NYLON TM , optical fiber bundles,
- the support is composed of a single material. In another embodiment, the support is composed of a mixture of several different materials.
- the support used in the present invention may be tubes, beads, slides, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, microtiter plates such as 768-well, 384-well, 96-well, 48-well, 24-well, 12-well, 8-well, 6-well, 4-well, 1-well, square grids, checkerboard grids, hexagonal arrays and the like.
- the support may be biological, non-biological, organic, inorganic, or any combination thereof.
- the surface of the support can be modified according to methods known to the skilled artisan, to promote trapping or immobilization of biological units and/or barcode units thereon.
- the trapping or immobilization of a biological unit and/or of a barcode unit to the support is aspecific.
- biological units and/or barcode units are trapped or immobilized in a layer of hydrogel that coats the support.
- the trapping or immobilization of a biological unit and/or of a barcode unit to the support is specific.
- the support comprises reactive groups on its surface, such as carboxyl, amino, hydroxyl, epoxy, and the like. In one embodiment, the support can have functional modifications, such as functional groups attached to its surface. In one embodiment, the support comprises at least one means involved with binding at least one biological unit and/or at least one barcode unit.
- Means for binding a biological unit and/or a barcode unit comprise, but are not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, and a hydrophobic group, as described hereinabove.
- the coating may be an all-over coating, i.e., completely covering the support, or may be a partial coating, i.e., covering only parts of the support.
- coating of a support with a means for binding a biological unit and/or a barcode unit requires functionalization of the support.
- functionalized supports include, but are not limited to, amino-functionalized supports, carboxyl-functionalized supports, hydroxyl-functionalized supports, and epoxy-functionalized supports.
- Techniques to functionalize a support are well-known in the art and include, but are not limited to, organosilane crosslinking, such as methoxysilane, ethoxysilane and acetoxysilane derivatives.
- Examples of techniques for coating a support with a means for binding a biological unit and/or a barcode unit include, but are not limited to, adsorption and covalent attachment.
- Covalent attachment may be performed on functionalized supports, using coupling agents such as carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfo-NHS, dimethylaminopropyl (DEAP), glutaraldehyde, aldehyde, sodium cyanoborohydride (NaCNBH 3 ), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), dithiothreitol (DTT), and/or cyanogen bromide (BrNC).
- EDC carbodiimide
- NHS N-hydroxysuccinimide
- sulfo-NHS dimethylaminopropyl
- DEP dimethylaminopropyl
- SPDP succinimidyl 3-(2-pyridyldithi
- each barcode unit comprises at least a means involved with binding a biological unit as defined hereinabove.
- each biological unit comprises at least a means involved in binding the barcode unit as defined hereinabove.
- each barcode unit comprises a unique barcode as defined hereinabove. In one embodiment, each barcode unit comprises clonal copies of a unique barcode.
- each barcode unit comprises at least one nucleic acid sequence primer as defined hereinabove.
- each barcode unit comprises a nucleic acid oligonucleotide as defined hereinabove.
- the plurality of biological units is bound to a support as defined hereinabove.
- the plurality of barcode units is bound to a support as defined hereinabove.
- the methods according to the present invention may comprise a step of selection and/or sorting of the biological units.
- Selection and/or sorting of biological units may be based on the expression of a given surface molecule such as a protein or a carbohydrate, or on specific light scattering and fluorescence characteristics of each biological unit. Selection and/or sorting of biological units may also be bases on their size.
- Methods to select and/or sort biological units are well-known to the skilled artisan, and comprise, but are not limited to, fluorescent activated cell sorting (FACS), fluorescence in situ hybridization-flow cytometry (FISH-FC), IsoRaft array, DEPArray lab-on-a-chip technology, magnetic cell sorting, immunoprecipitation, filtration and the like.
- the methods according to the present invention may comprise a step of lysis of the biological units.
- the methods according to the present invention may comprise a step of reverse transcription of the biological units' RNA content, preferably of the biological units' mRNA content.
- biochemistry and molecular biology assays can be carried out before, during or after the step of barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- the methods according to the present invention may comprise a step of pre-amplification of the biological units' nucleic acids, such as DNA, RNA or cDNA. In one embodiment, the methods according to the present invention may comprise a step of pre-amplification of the biological units' nucleic acids, such as DNA, RNA or cDNA, before the step of barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- the methods according to the present invention may comprise a step of purifying templates for biochemistry and molecular biology assays. Endogenous or exogenous proteins and complexes bound to nucleic acid templates or membranes encapsulating nucleic acid templates can be removed from the hydrogel after biological unit/barcode unit complex trapping. Techniques for nucleic acid purification are well known to the skilled artisan and include, without limitation, the use of proteinase K and/or detergents such as SDS, sarkosyl, NP-40, and the like.
- the methods according to the present invention may comprise a step of cleaning amplified nucleic acids.
- a nucleic acid library for sequencing Prior to preparing a nucleic acid library for sequencing, it can be desirable to remove single-stranded primers and reaction products such as enzymes.
- Techniques for nucleic acid clean-up are well known to the skilled artisan, and include without limitation, the use of single-strand-specific nucleases and/or the use of phosphatases to dephosphorylate phosphorylated ends of nucleic acids.
- single-strand-specific nucleases include, but are not limited to, exonuclease I, mung bean nuclease, nuclease Bh1, nuclease P1, nuclease S1, BAL 31 nuclease.
- phosphatases include, but are not limited to, alkaline phosphatase such as shrimp alkaline phosphatase.
- the methods according to the present invention may comprise a step of sizing the amplified nucleic acids.
- Short-read sequencers such as Illumina or Ion Torrent, operate best when fed DNA libraries that contain fragments of similar sizes, according to the manufacturer's recommendations. When libraries are not properly size-selected, these sequencers can become less efficient.
- Techniques for DNA size selection are well known to the skilled artisan, including, but not limited to, nucleic acid gel electrophoresis, bead-based protocols, pulsed-field gel electrophoresis (PFGE), automated size selection.
- PFGE pulsed-field gel electrophoresis
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library fragmentation.
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library enzymatic fragmentation.
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library mechanical fragmentation.
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library polishing.
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library A-tailing.
- the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library ligation.
- the methods according to the present invention may comprise a step of tagmentation.
- Techniques for tagmenting nucleic acids and/or cDNA library are well known to the skilled artisan.
- the methods according to the present invention may comprise a step of nucleic acid sequencing.
- the sequencing of nucleic acids may be carried out by next generation sequencing (NGS).
- NGS next generation sequencing
- Methods for NGS of nucleic acid libraries are known to the skilled artisan, and comprise, but are not limited to, paired-end sequencing, sequencing by synthesis, and single-read sequencing.
- the methods according to the present invention comprise contacting the hydrogel matrix with biochemistry and molecular biology reagents, useful to carry out the method.
- the hydrogel matrix is porous enough to allow diffusion of biochemistry and molecular biology reagents, without allowing diffusion of the barcode unit, biological unit and/or analytes, such as for example, nucleic acids extracted or derived from a discrete biological unit.
- subsequent steps can be performed by exchanging and/or washing biochemistry and molecular biology reagents in contact with the hydrogel matrix.
- Biochemistry and molecular biology reagents are well-known to the skilled artisan, and encompass all reagents known to perform biochemistry and molecular biology assays, such as solutions (buffer solutions, wash solutions, and the like), detergents, enzymes, nucleic acid primers, and the like.
- diffusion of biochemistry and molecular biology reagents is a passive diffusion.
- Passive diffusion includes, but is not limited to, osmosis and diffusiophoresis.
- diffusion of biochemistry and molecular biology reagents is an active diffusion.
- Techniques for active diffusion in a hydrogel are well-known to the skilled artisan, and include, but are not limited to, the use of pumps, electroosmosis and electrophoresis.
- subsequent steps are performed by exchanging the majority reagent in contact with the hydrogel.
- the methods according to the present invention do not require the use of expensive oils, chips and/or droplet generation instruments.
- the methods according to the present invention can be automated.
- the methods according to the present invention may comprise a step of dissolving the hydrogel matrix.
- dissolving of the hydrogel matrix can occur at any time throughout the method.
- Techniques to dissolve a hydrogel matrix are well-known to the skilled artisan, and comprise, but are not limited to, enzymatic depolymerization using enzymes such as agarase and thermal depolymerization using heat.
- dissolving of the hydrogel matrix can occur once at least one copy, preferably clonal copies of a unique barcode from at least one barcode unit have been incorporated into the biological unit and/or analytes, such as for example, nucleic acids extracted or derived from a discrete biological unit.
- depolymerization of the hydrogel matrix can occur once at least one nucleic acid extracted or derived from a discrete biological unit has primed to the at least one oligonucleotide, preferably to the at least one oligonucleotide comprising a nucleic acid sequence primer from a discrete barcode unit.
- the methods described herein can be implemented in a variety of applications, including, but not limited to, single-cell transcriptome profiling, single-cell genotyping, phasing, and single-cell epigenome profiling.
- the present invention relates to a method for analyzing gene expression in discrete biological units.
- Single-cell transcriptome profiling relies on the amplification of a single cell's mRNAs content and its sequencing.
- the generation of a single cell transcriptome generally requires a first step of reverse transcription to convert the mRNAs with poly(A) tails into first-strand cDNAs, which can be further amplified and sequenced.
- the method for analyzing gene expression in discrete biological units may comprise the steps of:
- the method for analyzing gene expression in discrete biological units comprises additional steps which are well-known to the skilled artisan. Such steps are described in Macosko et al., 2015. Cell. 161:1202-1214 ; Fan et al., 2015. Science. 347(6222):1258367 ; Klein et al., 2015. Cell. 161(5):1187-201 ; Gierahn et al., 2017. N ⁇ t Methods. 14(4):395-398 ; and US patent applications US2016-0289669 , US2016-0265069 , US2016-0060621 and US2015-0376609 .
- each barcode unit comprises at least one oligonucleotide comprising a poly-dT nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- each barcode unit comprises at least one oligonucleotide comprising a poly-dU nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- each barcode unit comprises at least one oligonucleotide comprising a (dT)nVN nucleic acid sequence primer, a unique barcode and/or a PCR handle, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e. A, C or G) and N represents any nucleotide (i.e. A, T/U, C or G).
- each barcode unit comprises at least one oligonucleotide comprising a (dU)nVN nucleic acid sequence primer, a unique barcode and/or a PCR handle, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e. A, C or G) and N represents any nucleotide (i.e. A, T/U, C or G).
- releasing nucleic acids from each biological unit is performed by cell lysis, preferably by cell lysis using a non-ionic detergent and/or proteinase K.
- the method further comprises a step of washing out the non-ionic detergent and/or proteinase K.
- the method further comprises a step of inactivating proteinase K.
- inactivation of proteinase K is performed by heat and/or chemical inhibition.
- synthetizing a cDNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- synthetizing a cDNA library from the nucleic acids from each biological unit is performed by reverse transcription, i.e ., using a reverse transcriptase.
- the reverse transcriptase is a M-MLV reverse transcriptase.
- a complementary strand of the cDNAs of the cDNA library is synthetized, preferably using second strand reaction components.
- the complementary strand of the cDNAs of the cDNA library is synthetized using RNAse H, DNA polymerase I and/or DNA ligase.
- the cDNA library is fragmented, to obtain cDNA fragments.
- Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- cDNA fragments are polished. In one embodiment, cDNA fragments are A-tailed.
- adaptors are added to the cDNA library.
- Adaptors may be added to the cDNA library using various methods, including but not limited to, Tn5 transposition and ligation.
- amplification of the cDNA library is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- the present invention also relates to a method for analyzing the genotype in discrete biological units.
- Single-cell genotyping relies on the whole genome amplification (WGA) of a single cell's DNA to generate enough DNA for sequencing.
- WGA whole genome amplification
- Several methods for WGA are available and well-known to the skilled artisan. Some methods however lead to amplification bias, and subsequent inadequate genome coverage.
- PCR-based exponential WGA with degenerate primers introduces sequence-dependent bias.
- MDA Multiple displacement amplification
- MALBAC Multiple annealing and loop-based amplification cycles
- the method for analyzing the genotype in discrete biological units may comprise the steps of:
- the method for analyzing the genotype in discrete biological units comprises additional steps which are well-known to the skilled artisan. Such steps are described in Hutchison et al., 2005. Proc Natl Acad Sci USA. 102(48):17332-6 ; Leung et al., 2016. Proc Natl Acad Sci USA. 113(30):8484-9 ; Wang et al., 2012. Cell. 150(2):402-12 ; Marcy et al., 2007.
- PLoS Genet. 3(9):1702-8 Gole et al., 2013. Nat Biotechnol. 31(12):1126-32 ; Zhang et al., 2006. Nat Biotechnol. 24(6):680-6 ; and International applications WO2016/061517 and WO2005/003304 .
- each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- each barcode unit comprises at least one oligonucleotide comprising an oligo-dN primer (such as an hexanucleotide d(N 6 ) or an octanucleotide d(N 8 ) primer, wherein N represents any nucleotide ( i.e., A, T/U, C or G)), a unique barcode and/or a PCR handle.
- an oligo-dN primer such as an hexanucleotide d(N 6 ) or an octanucleotide d(N 8 ) primer, wherein N represents any nucleotide ( i.e., A, T/U, C or G)
- releasing genomic DNA from each biological unit is performed by cell and/or nucleus lysis, preferably by cell and/or nucleus lysis using an ionic detergent and/or proteinase K.
- the method further comprises a step of washing out the ionic detergent and/or proteinase K.
- the method further comprises a step of inactivating proteinase K.
- inactivation of proteinase K is performed by heat and/or chemical inhibition.
- the method further comprises a step of denaturation of the genomic DNA.
- Methods to denature genomic DNA are well-known to the skilled artisan and include, but are not limited to, alkaline treatment and/or heat.
- synthetizing a cDNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- synthetizing a cDNA library from the nucleic acids from each biological unit is performed by primer-directed extension.
- amplification of genomic DNA is performed by whole genome amplification (WGA). In one embodiment, amplification of genomic DNA is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- amplified genomic DNA is fragmented, to obtain DNA fragments.
- Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- cDNA fragments are polished. In one embodiment, cDNA fragments are A-tailed.
- adaptors are added to the DNA fragments.
- Adaptors may be added to the DNA fragments using various methods, including but not limited to, Tn5 transposition and/or ligation.
- the method for analyzing the genotype in discrete biological units may implement direct library preparation (DLP).
- DLP direct library preparation
- amplified genomic DNA is tagmented.
- unamplified genomic DNA is tagmented.
- Direct library preparation and tagmentation are well-known to the skilled artisan. Reference can be made, e.g., to Vitak et al., 2017. Nat Methods. 14(3):302-308 ; Adey et al., 2010. Genome Biol. 11(12):R119 ; Gertz et al., 2012. Genome Res. 22(1):134-41 ; and Zahn et al., 2017. N ⁇ t Methods. 14(2):167-173 .
- each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor.
- the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2).
- the method comprises a step of ligating the tagmented genomic DNA from each biological unit to the at least one oligonucleotide of each barcode unit.
- the method comprises a step of amplification of the DNA fragments.
- Techniques to amplify DNA fragments are well-known to the skilled artisan.
- amplification of the DNA fragments is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplification of the DNA fragments is performed with at least one nucleic acid sequence primer which is not the at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- the present invention also relates to a method for analyzing the haplotype of discrete biological units, i.e., for phasing.
- Phasing relies on the whole genome amplification (WGA) of a high molecular weight, i.e., greater than 25 or 50 kilobases, DNA to generate enough DNA for sequencing.
- WGA whole genome amplification
- Several methods for WGA are available and well-known to the skilled artisan. Some methods however lead to amplification bias, and subsequent inadequate genome coverage.
- PCR-based exponential WGA with degenerate primers introduces sequence-dependent bias.
- MDA Multiple displacement amplification
- MALBAC Multiple annealing and loop-based amplification cycles
- the Tn5 transposase and subsequent amplification can be used for library prep in a method termed "Contiguity-Preserving Transposition" (CPT-seq) ( Amini et al., 2014. N ⁇ t Genet. 46(12):1343-9 ).
- CPT-seq Contiguity-Preserving Transposition
- the first step after the genomic DNA has been optionally purified is to tagment the DNA through Tn5 transposition. This fragments the DNA and adds universal adaptors directly to the template. After gap filling, PCR then occurs using primers complementary to the inserted Tn5 adaptors followed by sequencing.
- the method for analyzing the haplotype of discrete biological units may comprise the steps of:
- the method for analyzing the haplotype in discrete biological units comprises additional steps which are well-known to the skilled artisan. Such steps are described in International applications WO2015/126766 , WO2016/130704 , WO2016/61517 , WO2015/95226 , WO2016/003814 , WO2005/003304 , WO2015/200869 , WO2014/124338 , WO2014/093676 ; US patent application US2015-066385 ; Kuleshov et al., 2014. N ⁇ t Biotechnol. 32(3):261-6 ; Amini et al., 2014. Nat Genet. 46(12):1343-9 ; Kaper et al., 2013.
- the at least one means for binding a biological unit is an anti-Tn5 antibody. In one embodiment, the at least one means for binding a biological unit is streptavidin and the biological unit is contacted with a biotinylated anti-Tn5 antibody.
- each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor.
- the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2).
- each barcode unit comprises at least one oligonucleotide comprising an oligo-dN primer (such as an hexanucleotide d(N 6 ) or an octanucleotide d(N 8 ) primer, wherein N represents any nucleotide ( i.e., A, T/U, C or G)), a unique barcode and/or a PCR handle.
- oligo-dN primer such as an hexanucleotide d(N 6 ) or an octanucleotide d(N 8 ) primer, wherein N represents any nucleotide ( i.e., A, T/U, C or G)
- releasing nucleic acids from each biological unit is performed by cell and/or nucleus lysis, preferably by cell and/or nucleus lysis using an ionic detergent and/or proteinase K.
- synthetizing a DNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- the method further comprises a step of washing out the ionic detergent and/or proteinase K.
- the method further comprises a step of inactivating proteinase K.
- inactivation of proteinase K is performed by heat and/or chemical inhibition.
- the method further comprises a step of denaturation of the nucleic acids from each biological unit.
- Methods to denature nucleic acids are well-known to the skilled artisan and include, but are not limited to, alkaline treatment and/or heat.
- amplification of the nucleic acids from each biological unit is performed by whole genome amplification (WGA). In one embodiment, amplification of the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplified nucleic acids from each biological unit are fragmented, to obtain nucleic acid fragments. Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- nucleic acid fragments are polished. In one embodiment, nucleic acid fragments are A-tailed.
- adaptors are added to the nucleic acid fragments, preferably Tn5 adaptors.
- Adaptors may be added to the nucleic acid fragments using various methods, including but not limited to, Tn5 transposition and ligation.
- the method for analyzing the haplotype in discrete biological units may implement contiguity-preserving transposition (CTP-seq). Such method is described in international application WO2016/061517 .
- the method comprises a step of tagmenting nucleic acids from each biological unit, preferably with Tn5 transposase.
- nucleic acids from each biological unit are high molecular weight DNA (HMW-DNA).
- tagmenting HMW-DNA from each biological unit preserves the contiguity of the HMW-DNA from each biological.
- the method comprises a step of disrupting contiguity of the nucleic acids from each biological unit, preferably of the HMW-DNA from each biological unit.
- Techniques to disrupt contiguity are well-known to the skilled artisan and include, but are not limited to, release of Tn5 complexes from the nucleic acids from each biological unit, preferably by using an ionic detergent and/or proteinase K.
- the method comprises a step of gap filling of the adaptor, preferably of the Tn5 adaptor. In one embodiment, the method comprises a step of amplification of the tagmented nucleic acids from each biological unit. In one embodiment, amplification of the tagmented nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, the method comprises a step of ligating the tagmented nucleic acids from each biological unit to the at least one oligonucleotide of each barcode unit.
- the method comprises a step of amplification of the tagmented nucleic acids.
- Techniques to amplify of nucleic acids are well-known to the skilled artisan.
- amplification of the tagmented nucleic acids is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- the present invention also relates to a method for analyzing the epigenome in discrete biological units.
- Single cell nucleosome positioning based on Tn5 transposition has been developed, termed “Assay for Transposase-Accessible Chromatin with high throughput sequencing” (ATAC-seq) ( Buenrostro et al., 2015. Nature. 523(7561):486-90 ).
- the first step enable molecular access to nucleosome-free DNA by using low percentage non-ionic detergents on intact cells or isolated nuclei.
- the accessible DNA is then tagmented through Tn5 transposition. This fragments the DNA and adds universal adaptors directly to the template. PCR then occurs using primers complementary to those adaptors followed by sequencing.
- the method for analyzing the epigenome in discrete biological units may comprise the steps of:
- non-nucleosome start sites are sites where transposition occurs, i.e., where the DNA is accessible.
- non-nucleosome start sites are sites where DNA is enzymatically fragmented and where DNA is ligated.
- the method for analyzing the epigenome in discrete biological units comprises additional steps which are well-known to the skilled artisan. Such steps are described in International application WO2014/189957 ; Buenrostro et al., 2015. Nature. 523(7561):486-90 ; Buenrostro et al., 2013. N ⁇ t Methods. 10(12):1213-8 ; and Christiansen et al., 2017. Methods Mol Biol. 1551:207-221 .
- each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- the nucleic acid sequence primer has a sequence which is complementary to at least one adaptor sequence, preferably at least one Illumina adaptor sequence.
- the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor.
- the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2).
- releasing non-nucleosome bound DNA from each biological unit is performed by cell lysis, preferably by cell lysis using a non-ionic detergent and/or proteinase K.
- synthetizing a DNA library from the non-nucleosome bound DNA from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- the method further comprises a step of washing out the non-ionic detergent and/or proteinase K.
- the method further comprises a step of inactivating proteinase K.
- inactivation of proteinase K is performed by heat and/or chemical inhibition.
- non-nucleosome bound DNA is tagmented. Techniques for tagmentation are well-known to the skilled artisan. In one embodiment, tagmentation of non-nucleosome bound DNA is performed by Tn5 transposition, preferably using Illumina adaptor sequences.
- the method comprises a step of ligating the tagmented non-nucleosome bound DNA from each biological unit to the at least one oligonucleotide of each barcode unit.
- the method comprises a step of amplification of the tagmented non-nucleosome bound DNA from each biological unit.
- Techniques to amplify DNA are well-known to the skilled artisan.
- amplification of the tagmented non-nucleosome bound DNA is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplification of the tagmented non-nucleosome bound DNA is performed with at least one nucleic acid sequence primer which is not the at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- amplification of the tagmented non-nucleosome bound DNA incorporates the adaptor sequence from the Tn5 transposases into the amplification products from each biological unit.
- amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- Example 1 Trapping and barcoding discrete biological units in a hydrogel
- the present invention relates to the trapping of discrete biological units (i.e., cells or groups of cells, viruses, organelles, macromolecular complexes or biological macromolecules).
- discrete biological units i.e., cells or groups of cells, viruses, organelles, macromolecular complexes or biological macromolecules.
- biological unit/barcode unit complexes are formed, each complex comprising a single barcode unit and a single biological unit (step 1 of Figure 1 ).
- Biological unit/barcode unit complexes can be formed upon binding and/or immobilization of the biological unit on the barcode unit.
- Barcode units must thus carry on their surface a means for binding, either specifically or non-specifically, biological units. These means include proteins or fragments thereof, peptides, antibodies or fragments thereof, nucleic acids, carbohydrates, vitamins or derivatives thereof, coenzymes or derivative thereof, receptor ligands derivative thereof and/or hydrophobic groups.
- the biological units must carry, either naturally or not, a complementary means, binding to the means of the barcode unit.
- a means for binding a biological unit can be an antibody, directed to molecules expressed or present (either naturally or artificially) at the surface of the biological unit.
- Another option can be the use of a biotinylated antibody directed to molecules expressed or present at the surface of the biological unit, and the subsequent binding of the biological unit carrying the biotinylated antibody to barcode units coated with streptavidin.
- the biological unit/barcode unit complexes can be contacted with a hydrogel solution, which upon polymerization, traps the biological unit/barcode unit complexes (steps 2-3 of Figure 1 ). Biochemistry and molecular biology assays can then be performed directly in the hydrogel matrix, by contacting the hydrogel with any required reagent and/or solution.
- a suitable hydrogel solution can be alginate. Its fine grain size allows for the formation of very small pores upon polymerization with calcium, trapping the biological unit/barcode unit complexes without any risk of diffusion, while still allowing for the diffusion of smaller components like reagent and/or solution.
- a first step will comprise the lysis of the biological unit, to release its nucleic acid content. Any detergent level is supported by the hydrogel platform, allowing to lyse even difficult-to-lyse biological units.
- each barcode unit comprises clonal copies of an oligonucleotide, which is composed of at least one priming site (nucleic acid sequence primer) and a barcode sequence.
- the barcode sequence should always be identical in every oligonucleotide of a given barcode unit, so as to allow identification of the source or origin of the nucleic acids extracted or derived from one discrete biological unit.
- barcoding i.e., priming of the biological unit's nucleic acids to the barcode unit's nucleic acid sequence primer
- classical biochemistry and molecular biology assays can be carried out on the barcoded nucleic acids, either while still entrapped in the hydrogel matrix, or in solution, after hydrogel matrix has been dissolved.
- primer-directed extension ligation, amplification, fragmentation, addition of adaptor sequences, next generation sequencing and the like (steps 5-6 of Figure 1 ).
- alginate as a hydrogel
- calcium can be washed out from the hydrogel to allow depolymerization.
- Stabilization of the primed, i.e., barcoded nucleic acids, prior to any biochemistry and molecular biology assay, and in particular, prior to primer-directed extension can be achieved using other cations, such as sodium.
- a crucial step when implementing the method of the present invention is the binding of a single biological unit to a single barcode unit, as to form a 1:1 complex.
- the binding of multiple biological units to a single barcode unit skews the subsequent data retrieved, and in particular, single cell next generation sequencing data.
- sequences with "barcode 1" would be biased or corrupted since they are gathered from two distinct biological units.
- Figure 4 shows the immobilization of the biological units of interest on a support, coated with means for binding said biological units (step 11).
- biological units can be contacted with barcode units (step 12) - preferentially with barcode units which are larger in size with respect to the biological units, to create hindrance and prevent the binding of multiple barcode unit on a single biological unit (step 1). Therefore, since only one barcode unit is bound per biological unit, it is possible to parse subsequent next generation sequencing data into single biological units.
- Such configuration can be easily implemented, using a support such as a microcentrifuge tube coated with a means for binding biological units, such as biotin.
- biological units such as cells are contacted with streptavidin-coupled antibodies, then deposited in the tube to allow for binding. Excess cells are removed.
- Biotin-coated barcode units such as beads, are then deposited in the tube to allow for binding to the cells. Excess beads are removed.
- a hydrogel solution is then poured into the tube, such as sodium alginate, together with calcium ions, to allow alginate to polymerize. Trapped cells can then be processed, such as for example by addition of detergent on top of the tube.
- the detergent By capillarity, the detergent reaches the trapped cells and lyse their membrane, releasing their nucleic acid content.
- Alginate pore size is small enough to avoid diffusion of nucleic acids, while allowing diffusion of smaller reactants and substrates. Barcoding occurs as nucleic acids from a discrete cell are released and attach to the nucleic acid sequence barcode of their adjacent barcode bead. Once the nucleic acids are properly barcoded, the sample can be wash out to remove calcium ions. Alginate hydrogel dissolves, and further steps can be processed directly in the tube, in solution.
- barcode units can be bound on a support, coated with means for binding said barcode units. Once bound to the support, barcode units can be contacted with biological units - preferentially with biological units which are larger in size with respect to the barcode units, to create hindrance and prevent the binding of multiple biological units on a single barcode unit ( Figure 5 ).
- Such configuration can also be implemented using a support such as a microcentrifuge tube coated with a thin layer of hydrogel which, upon polymerization, immobilizes barcode units throughout the support.
- Biological units such as cells are then deposited in the tube to allow for binding to the barcode units (providing that the layer of hydrogel immobilizing the barcode units is thinner than the smallest dimension of the barcode unit, i.e., that at least a part of the barcode unit remains accessible for contacting biological units). Excess cells are removed.
- a hydrogel solution is then poured into the tube and left polymerizing. Trapped cells can then be processed as described hereinabove. Once the nucleic acids are properly barcoded, both hydrogels (i.e., the thin layer coating the tube and the hydrogel matrix trapping the biological units) can be dissolved, and further steps can be processed directly in the tube, in solution.
- Another strategy to avoid the formation of non-stoichiometric biological unit/barcode unit complexes is the use of a support where biological units of interest ( Figure 6 ) or barcode units ( Figure 7 ) are bound and/or immobilized as described previously, together with limiting concentrations of barcode units or biological units, respectively.
- concentration of free units is lower than the concentration of support-bound units (biological units or barcode units, respectively). This ensures the binding of at most one barcode unit per biological unit and conversely, making it possible to parse subsequent next generation sequencing data into single biological units.
- Some biological units (step 1 of Figure 6 ) or barcode units (step 1 of Figure 7 ) are not coupled with a barcode unit or a biological unit, respectively, and therefore do not produce any data.
- Single-cell transcriptome profiling is one of the numerous biochemistry and molecular biology assays that can be carried out using the method of the present invention ( Figure 8 ).
- the biological units will be a cell, such as a mammalian cell for example, or any other cell suitable for single-cell transcriptome profiling.
- Single-cell transcriptome profiling relies on the amplification of a single cell's mRNAs content and its sequencing.
- a first step is therefore to release the cells' mRNAs content, by lysing the cells directly in the hydrogel.
- non-ionic detergents or any other suitable reagent for cell lysis can be applied directly on the hydrogel matrix. By diffusion, the reagent can reach up to the biological units, and lyse them (step 83* of Figure 8 ) .
- the released mRNAs bind in their local environment to the oligonucleotides carried by the barcode units.
- These oligonucleotides are present in multiple clonal copies on each barcode unit, and are unique as to their sequence from barcode unit to barcode unit. They comprise a PCR handle, a unique barcode sequence and a nucleic acid sequence primer.
- priming i.e ., barcoding
- first-strand cDNA synthesis will occur in 3' of the barcode unit oligonucleotide, using a reverse transcriptase enzyme.
- Second strand cDNA synthesis can then occur, optionally through template switching and amplification (step 85 of Figure 8 ).
- Next steps comprise for example fragmentation of the cDNA library, adaptering, and amplification.
- Barcoded, amplified and adaptered products can finally be sequenced by next generation sequencing (step 86 of Figure 8 ).
- Phasing is another molecular biology assay that can be carried out using the method of the present invention ( Figure 9 ) .
- transposomes are assembled in solution by mixing a Tn5 transposase with high molecular weight DNA (i.e., the biological unit).
- This step sometimes referred to as tagmentation, creates contiguity preserved transposition DNA (CPT-DNA) fragments, and is followed by a second step wherein the transposomes are contacted with barcode units, comprising a means for binding the biological unit (step 91 of Figure 9 ).
- this means binds Tn5 transposases.
- the CPT-DNA/barcode unit complexes are then contacted with a hydrogel solution, which is left to polymerize (steps 2-3 of Figure 9 ) .
- a hydrogel solution which is left to polymerize (steps 2-3 of Figure 9 ) .
- the Tn5 transposases are released, using ionic detergents and/or proteinase K, disrupting thus contiguity and yielding DNA fragments comprising a Tn5 adaptor sequence (step 94 of Figure 9 ) .
- the released DNA fragments comprising a Tn5 adaptor sequence
- Tn5 adaptor sequence such as, e.g., SEQ ID NO: 1 or SEQ ID NO: 2.
- These oligonucleotides are present in multiple clonal copies on each barcode unit, and are unique as to their sequence from barcode unit to barcode unit. They comprise a PCR handle, a unique barcode sequence and a nucleic acid sequence primer, complementary to the Tn5 adaptor sequence (Tn5 adaptor primer, Tn5 P ).
- the following molecular biology steps can take place either within the hydrogel matrix or in solution, upon dissolving of the hydrogel.
- Ligation, gap-filling and amplification (step 95 of Figure 9 ), can occur either in the hydrogel matrix or in solution.
- Barcoded, amplified and adaptered products can finally be sequenced by next generation sequencing (step 96 of Figure 9 ).
Description
- The present invention relates to methods for trapping and barcoding discrete biological units in a hydrogel. In particular, the present invention relates to methods for discrete biological units' expression analysis. The methods of the present invention can further be used for single-cell transcriptome profiling, genotyping, phasing and/or haplotyping.
- To derive Next Generation Sequencing (NGS) analysis, three tasks must occur: 1) sample preparation (sample prep), 2) sequencing and 3) bioinformatics. Microfluidics has been exploited to improve the first of the three requirements, sample prep, specifically by enabling high throughput (HT) parallelization of reactions and efficiencies of scale. One application that has an acute need for HT microfluidic sample prep is single cell gene expression analysis by RNA sequencing (single cell RNAseq). The reason for this is that the number of cells to be analyzed can range from hundreds to thousands and each workflow starts by first isolating single cells in individual reaction chambers. Thus, the HT parallelization reaction capacity of any microfluidic platform needs to match these cell number requirements.
- The first microfluidic platform to be commercialized for single cell RNAseq analysis was based on PDMS (polydimethylsiloxane) chip technology. Available versions of the platform are able to process tens to hundreds of cells. Cells from a suspension are isolated in nanolitre (nL) volume PDMS chambers and then lysed by the application of a lysis reagent through the opening of valve and access to the appropriate lysis reagent inlet. Valve opening and selection of specific reagent inlets are done at each subsequent step to consecutively achieve reverse transcription of the mRNA, adaptor sequence addition to the cDNA and PCR. Amplicons from single cell products are then harvested from the chip and processed in bulk to finish sample prep from sequencing. Platforms that use PDMS architecture are limited since they require expensive multilayer PDMS chips and sophisticated pressure and thermal control instrumentation to operate those chips. Moreover, the number of reactions is determined by the smallest PDMS features that can be manufactured. For a reasonably sized chip, this means that no more than 1000 cells can be processed at a given time, which for a large proportion of biological samples, is not sufficient. And even if the throughput is adequate, PDMS infrastructure both from the chip and instrument perspective are prohibitively expensive.
- Water in oil droplet emulsions are another form of microfluidics. Compared to PDMS based technology, droplets have the advantage of providing a significant increase in reaction numbers. Throughput is only limited by the emulsion volume and the numbers increase proportionally with decreasing droplet size.
WO2016130704 andWO2017075265 have also disclosed methods based on hydrogel encapsulation of single cells. However, no barcoded particle that is able to form a complex with the cell was taught. Rather, barcoded particles were contacted with the cell after cell lysis. - The discovery that encapsulating beads coated with clonal oligos that are unique to each bead has enabled parallel molecular encoding of droplet reactions. For example, within the gene expression application space, after cell lysis in droplets, the bead oligos bind to the mRNA and in the process, encode a single cell transcriptome with a common bead molecular tag, otherwise known as a bead barcode. After sequencing, sequences with the same barcode can be grouped together, which effectively reconstructs the prior coupling of a bead and a cell in individual reaction chambers or droplets and enables single cell analysis. The molecular biology steps vary according to various forms of droplet encoding technological platforms. In Drop-SEQ, after binding of the mRNA to the bead oligos in droplets, reverse transcription and subsequent sample preparation steps take place in bulk on broken emulsions. In other commercially available platforms, reverse transcription occurs in droplets, with final sample preparation steps taking place in bulk.
- For example,
WO2015200541 discloses methods of analyzing nucleic acid of a plurality of cells, by forming partitions in a microfluidic device, each partition comprising a single barcoded particle and a single cell. Upon cell lysis, the capture sequence of the barcoded particle can hybridize to the nucleic acid of the single cell in each partition. - Although removing the throughput bottleneck of the PDMS chip technology, droplets have other significant drawbacks. First, droplets and their monodisperse formation are incompatible with detergent levels that are used to lyse difficult-to-lyse cells (such as plant cells, certain bacteria, in particular gram+ bacteria, molds, spores, yeasts, mycobacteria, etc.), access nuclei and perform a number of critical molecular biology steps. Second, performing multi-step molecular biology reactions is extremely difficult in droplets. Although possible through droplet merging or pico-injection, for example, multi-step droplet workflows significantly increases the complexity and cost of the microfluidic setup. Third, droplet platforms require high-grade oils, sophisticated chips whose features are difficult to manufacture at industrial scale, and instruments to accommodate and administer precise flow control through those chips. All three elements required for droplet platforms, namely oils, chips, and instruments, create a burden for manufacturing and tech support and, importantly, significantly increase the costs to the end user, thus limiting widespread droplet technology adoption.
- The current invention is designed to eliminate the drawbacks of the existing technologies. Indeed, the inventors have surprisingly developed a new method for single cell gene expression analysis, that does not require PDMS chips or droplets, while preserving the key benefit of droplet platforms in being able to process greater than thousands of cells. Based on the use of a hydrogel platform, this new technology also resolves the three key problems associated with droplet technologies. First, any detergent level is supported by the hydrogel platform, creating the possibility of lysing any cell or nuclei, as well as supporting key biochemistry and molecular biology reactions. Second, multistep reactions can be performed with ease since soluble reagents can easily access the reactor space through the hydrogel. Subsequent reactions are performed by simply exchanging the majority solution in contact with the hydrogel. Third, there is no need for expensive oils, chips and/or droplet generation instruments. For automation, an instrument may be used to manage the hydrogel reactor platform, but is not required.
- The limitations of PDMS and droplet technologies and the improvements of the hydrogel reactor platform are not restricted to the single cell gene expression space. They apply to any application where the substrate has multiple primer binding sites, such as single cell genomes and long naked DNA molecules that are used as substrates in phasing and genome structure applications. The molecular biology reactions vary according to the identity of the substrate and the output requirements of the sample prep method. However, the foundational methods to trap and barcode biological units in hydrogel remains unchanged.
- The present invention relates to a method for trapping discrete biological units in a hydrogel, said method comprising the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution, and
- c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,
- d) barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- The present invention further relates to a method for analyzing gene expression in discrete biological units, said method comprising the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing nucleic acids from each biological unit in the hydrogel matrix,
- e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,
- f) synthetizing a cDNA library from the nucleic acids from each biological unit,
- g) amplifying said cDNA library from each biological unit, wherein amplification of said cDNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, and
- h) optionally, sequencing the amplification products.
- The present invention further relates to a method for analyzing the genotype in discrete biological units, said method comprising the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing genomic DNA from each biological unit in the hydrogel matrix,
- e) barcoding said genomic DNA from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit,
- g) amplifying said genomic DNA or DNA library from each biological unit, wherein amplification of said genomic DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products of each biological unit, and
- h) optionally, sequencing the amplification products.
- The present invention further relates to a method for analyzing the haplotype of discrete biological units, said method comprising the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,
- d) optionally, releasing nucleic acids from each biological unit in the hydrogel matrix,
- e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit
- g) amplifying said nucleic acid or DNA library from each biological unit, wherein amplification of said nucleic acids or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, and
- h) optionally, sequencing the amplification products.
- The present invention further relates to a method for analyzing the epigenome in discrete biological units, said method comprising the steps of:
- a) contacting a plurality of cellular biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,
- e) barcoding said non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the non-nucleosome bound DNA from each biological unit,
- g) amplifying said non-nucleosome-bound-DNA or DNA library from each biological unit, wherein amplification of said non-nucleosome-bound-DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, and
- h) optionally, sequencing the amplification products.
- In one embodiment, the biological units are immobilized on a support. In one embodiment, the barcode units are immobilized on a support.
- In one embodiment, the biological units are immobilized on a support in a hydrogel layer. In one embodiment, the barcode units are immobilized on a support in a hydrogel layer.
- In one embodiment, the unique barcode is present in multiple clonal copies on each barcode unit.
- In one embodiment, the unique barcode comprises a nucleic acid sequence barcode.
- In one embodiment, the unique barcode comprises a nucleic acid sequence primer. In one embodiment, the nucleic acid sequence primer comprises random nucleic acid sequence primers. In one embodiment, the nucleic acid sequence primer comprises specific nucleic acid sequence primers.
- In one embodiment, the at least a means involved with binding biological units comprises proteins, peptides and/or fragments thereof; antibodies and/or fragments thereof; nucleic acids; carbohydrates; vitamins and/or derivatives thereof; coenzymes and/or derivatives thereof; receptor ligands and/or derivatives thereof; and/or hydrophobic groups.
- In one embodiment, each barcode unit consists of a bead.
- In one embodiment, the step of barcoding is carried out in the hydrogel matrix by primer template annealing.
- In one embodiment, the step of barcoding is carried out in the hydrogel matrix by primer-directed extension.
- In one embodiment, the step of barcoding is carried out in the hydrogel matrix by ligation.
- In one embodiment, discrete biological units comprise cells, groups of cells, viruses, nuclei, mitochondria, chloroplasts, biological macromolecules, exosomes, chromosomes, contiguity preserved transposition DNA fragments and/or nucleic acid fragments.
- In one embodiment, cells or groups of cells comprise cells in in vitro culture, stem cells, tumor cells, tissue biopsy cells, blood cells and tissue section cells.
- In the present invention, the following terms have the following meanings:
- The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" preceding a figure means plus or less 10% of the value of said figure. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.
- The term "amplification" refers to the process of producing multiple copies, i.e., at least 2 copies, of a desired template sequence. Techniques to amplify nucleic acids are well known to the skilled artisan, and include specific amplification methods as well as random amplification methods.
- The term "A-tailing" refers to an enzymatic method for adding a non-templated A nucleotide to the 3' end of a blunt, double-stranded DNA molecule.
- The term "barcode" refers to a molecular pattern which can be used as a unique identifier, to uniquely identify a discrete biological unit. The term "barcode" further refers to the molecular pattern which is used to identify the source or origin of an analyte within a sample, such as for example, a nucleic acid sequence extracted or derived from a discrete biological unit.
- The term "barcode unit" refers to an identifiable substrate or matrix upon which a biological unit can be bound or immobilized. The barcode unit may be rigid, solid or semi-solid.
- The term "barcoding" refers to the attachment of a discrete barcode unit's barcode, preferably a nucleic acid barcode, to the biological unit template nucleic acid sequences through primer template annealing, primer dependent DNA synthesis and/or ligation.
- The term "bead" refers to a discrete particle that may be spherical (e.g., microspheres) or have an irregular shape. Beads may be as small as about 0.1 µm in diameter or as large as about several millimeters in diameter.
- The term "biological unit" refers to discrete biological structures and portions, components or combinations of biological structures. Examples of biological units include, but are not limited to, a cell or a group of cells, a virus, an organelle such as a nucleus, a mitochondrion or a chloroplast, a macromolecular complex such as an exosome, a biological macromolecule such as a chromosome, a nucleic acid fragment, a contiguity preserved transposition DNA (CPT-DNA) fragment, a protein or a peptide.
- The term "carbohydrate" refers to any of a class of organic compounds with the general formula Cx(H2O)y. Carbohydrates include sugars, starches, celluloses, and gums. A carbohydrate may be a monosaccharide, a disaccharide, or a polysaccharide. Carbohydrates may be naturally occurring or synthetic.
- A monosaccharide is a monomer, or simple sugar, having a single chain or a single ring structure. Monosaccharides can be further classified by their structure and the number of carbon atoms in the ring or chain, such as aldoses, ketoses, pyranoses, furanoses, trioses, tetroses, pentoses, hexoses, and heptoses, among others. Examples of monosaccharides include, but are not limited to, N-acetylglucosamine, allose, altrose, arabinose, deoxyribose, dihydroxyacetone, erythrose, fructose, fucose, α-L-fucopyranose, galactose, β-D-galactopyranose, galacturonic acid, glucose (dextrose), glucuronic acid, glyceraldehyde, gulose, idose, lyxose, mannose, α-D-mannopyranose, mannuronic acid, neuraminic acid, psicose, rhamnose, ribose, ribulose, sorbose, tagatose, threose, xylose, and xylulose.
- Disaccharides are formed from two monosaccharides joined by glycosidic bonds. Examples of disaccharides include, but are not limited to, cellobiose, gentiobiose, isomaltose, lactose, lactulose, laminaribiose, maltose, mannobiose, melibiose, nigerose, rutinose, sucrose, trehalose, and xylobiose. Polysaccharides are polymers formed from two or more monosaccharides joined by glycosidic bonds. Polysaccharides formed from 3-10 monosaccharides are often called oligosaccharides. Examples of polysaccharides include, but are not limited to, agarose, alginate, amylopectin, amylose, carageenan, cellulose, chitin, chitohexanose, chitosan, chondroitin sulfate, curdlan, dermatan sulfate, dextran, dextrin, emulsan, furcellaran, galactomannan, glucomannan, gellan gum, glucosamine, glycogen, glycosaminoglycan, guar gum, gum arabic, heparan sulfate, heparin, hyaluronic acid, deacylated hyaluronic acid, inulin, isomaltulose, karaya gum, keratan sulfate, laminaran, locust bean gum, muramic acid, pectic acid, pectin, pullulan, pustulan, rhamsan gum, schizophyllan, scleroglucan, stachyose, starch, tragacant gum, welan gum, xanthan, and xanthan gum.
- As used herein, the term "carbohydrate" also refers to "glycoconjugates," which are carbohydrates covalently bonded to other chemical species such as, for example, proteins and lipids. Examples of glycoconjugates include, but are not limited to, glycolipids, glycopeptides, glycoproteins, lipopolysaccharides, and peptidoglycans.
- The term "cDNA library" refers to a library composed of complementary DNAs which are reverse-transcribed from mRNAs.
- The terms "cell" and "group of cells" include, but are not limited to, cells in in vitro culture; stem cells such as embryonic stem cells, adult stem cells, cancer stem cells, induced pluripotent stem cells or induced stem cells; tumor cells such as neoplastic cells; tissue biopsy cells; blood cells such as erythrocytes, leukocytes, mast cells, macrophages, thrombocytes or progenitor cells thereof; and tissue section cells.
- The term "clonal copies" refers to a population of identical copies of a single barcode.
- The terms "coat" and "coating" refer to the covering, modification or functionalization of a substrate, e.g., of a support and/or of a barcode unit.
- The term "coenzyme" refers to a non-protein element binding to an apoenzyme, which is a factor assisting an enzyme reaction by changing a chemical structure during an enzyme reaction and delivering functional elements such as atoms or electrons to a reaction substrate. The "coenzyme" may also be referred to as a "cofactor" or "helper enzyme".
- Examples of coenzymes include, but are not limited to, nicotinamide adenine dinucleotide (NAD), NADH, nicotinamide adenine dinucleotide phosphate (NADP), NADPH, adenosine triphosphate (ATP), phosphoadenylyl sulfate (PAPS), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine triphosphate (GTP), inosine triphosphate (ITP), thiamine pyrophosphate (TPP), flavin mononucleotide (EMM), flavin adenine dinucleotide (FAD), coenzyme-A (CoA), biocytin, tetrahydrofolic acid, coenzyme B12, lipoyllysine, 1,1-cis-retinal and 1,2,5-dihydroxycholecalciferol.
- The terms "complement" or "complementary" refer to a polynucleotide sequence capable of forming base pairing by hydrogen bonds with another polynucleotide sequence. For example, guanine (G) is the complementary base of cytosine (C), and adenine (A) is the complementary base of thymine (T) and of uracil (U).
- The term "contiguity" refers to a spatial relationship between two or more DNA fragments based on shared information. The shared aspect of the information can be with respect to adjacent, compartmental and distance spatial relationships. Information regarding these relationships in turn facilitates hierarchical assembly or mapping of sequence reads derived from the DNA fragments. This contiguity information improves the efficiency and accuracy of such assembly or mapping because traditional assembly or mapping methods used in association with conventional shotgun sequencing do not take into account the relative genomic origins or coordinates of the individual sequence reads as they relate to the spatial relationship between the two or more DNA fragments from which the individual sequence reads were derived.
- The term "copy of a desired template sequence" does not necessarily mean perfect sequence complementarity or identity to the template sequence. Copies can include, e.g., nucleotide analogs such as deoxyinosine, intentional sequence alterations and/or sequence errors that occur during amplification. In one embodiment, a copy of a desired sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100% identical to the template sequence.
- The term "detergent" refers to molecules having lipophilic as well as hydrophilic (i.e., amphiphilic) characteristics. Detergents are classified into four broad groupings, depending on the electrical charge of the surfactants:
- (1) Anionic detergents refer to detergents with a negative ionic charge. Examples of anionic detergents include, but are not limited to, sodium dodecyl sulfate (SDS), N-laurylsarcosine (sarcosyl), sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate and lithium dodecyl sulfate (LDS).
- (2) Cationic detergents refer to detergents with a positive ionic charge. Examples of cationic detergents include, but are not limited to, quaternary ammonium salts, amines with amide linkage, polyoxyethylene alkyl and alicyclic amines, N,N,N',N'tetrakis substituted ethylenediamines, 2-alkyl 1-
hydroxyethyl 2 imidazoline ethoxylated amines and alkyl ammonium salts. - (3) Non-ionic detergents refer to detergents which do not have any ionic groups. Examples of nonionic detergents include, but are not limited to, polysorbates, octylphenol ethoxylates, glucamines, Lubrol, Brij, Nonidet, poloxamers, Genapol and Igepal.
- Examples of polysorbates include, but are not limited to, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65 (Tween 65), polysorbate 80 (Tween 80) and polysorbate 85 (Tween 85).
- Examples of octylphenol ethoxylates include, but are not limited to, Triton X-15, Triton X-35, Triton X-45, Triton X-100, Triton X-102, Triton X-114, Triton X-165 (70%), Triton X-305 (70%), Triton X-405 (70%) and Triton X-705 (70%).
- Examples of glucamines include, but are not limited to, N-octanoyl-N-methylglucamine (MEGA-8), N-nonanoyl-N-methylglucamine (MEGA-9) and N-decanoyl-N-methylglucamine (MEGA-10).
- Examples of Lubrol include, but are not limited to, Lubrol WX, Lubrol PX, Lubrol 12A9, Lubrol 17A10, Lubrol 17A17, Lubrol N13 and Lubrol G.
- Examples of Brij include, but are not limited to, Brij 35, Brij 58, Brij 93, Brij 97, Brij C2, Brij S2, Brij L4, Brij C10, Brij O10, Brij S10, Brij 020, Brij S20, Brij L23 and Brij S100.
- Examples of Nonidet include, but are not limited to, Nonidet P40.
- Examples of poloxamer include, but are not limited to, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 184, poloxamer 188 (Pluronic F68), poloxamer 331, poloxamer 407 (Pluronic F127)
- Examples of Genapol include, but are not limited to, Genapol X-080, Genapol X-100 and Genapol C-100.
- Examples of Igepal include, but are not limited to, Igepal CA-210, Igepal CA-520, Igepal CA-630, Igepal CA-720, Igepal CO-520, Igepal CO-630, Igepal CO-720, Igepal CO-890 and Igepal DM-970. (4) Zwitterionic detergents refer to detergents which have ionic groups, but no net charge. Examples of zwitterionic detergents include, but are not limited to, amidosulfobetaines, alkylbetaines and ammonio propanesulfonates such as amidosulfobetaine-14, amidosulfobetaine-16, 3-[(3-cholamidopropyl)dimethylarmnonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), 3-(4-heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate (C7BzO), EMPIGEN® BB, 3-(N,N-dimethyloctylammonio)propanesulfonate inner salt, 3-(decyldimethylammonio)propanesulfonate inner salt, 3-(dodecyldimethylammonio)propanesulfonate inner salt, 3-(N,N-dimethylmyristylammonio)propanesulfonate inner salt, 3-(N,N-dimethylpalmitylammonio)propanesulfonate inner salt, 3-(N,N-dimethyloctadecylammonio)propanesulfonate inner salt.
- The term "epigenome" refers to all the chemical changes to the DNA and/or histone proteins of a cell, and responsible for gene expression regulation, development, differentiation and suppression of transposable elements.
- The term "genome structure" refers to the order, numbers and presence of genetic units (such as loci, genes and the like) positioned along a chromosome.
- The term "haplotype" refers to a group of genes from different loci on a single chromosome that are inherited together from a single parent. Haplotype information contributes to the understanding of the potential functional effects of gene variants on the same (in cis) or allelic (in trans) strand of DNA.
- The term "hydrogel" refers to a hydrophilic, high water-content, network of polymers, with physical or chemical crosslinks. Hydrogels are typically found in two states, depending among others on the extent of crosslinking: a sol state and a gel state. In the sol state, the hydrogel behaves as a liquid, while in the gel state, the hydrogel does not exhibit flow. As will clearly appear to the skilled person, while the hydrogel may already be a polymer in sol state, the terms "polymerizing the hydrogel" are used herein to designate the polymerization and/or crosslinking required to achieve sol to gel transition.
- The term "hydrogel matrix" refers to the physical structure of the hydrogel in gel state, i.e. the crosslinked network of polymers that achieves the desired porosity for the purpose of the invention, as further disclosed herein.
- The term "identity", when used in a relationship between the sequences of two or more nucleic acid sequences, refers to the degree of sequence relatedness between nucleic acids, as determined by the number of matches between strings of two or more nucleotide residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk, Arthur M. (1988), "Computational molecular biology", New York, NY: Oxford University Press; Smith, Douglas W. (1993), "Biocomputing: informatics and genome projects", New York, NY: Academic Press; Griffin, Annette M., and Hugh G. Griffin (1994), "Computer analysis of sequence data, ; vonHeinje, Gunnar (1987), "Sequence analysis in molecular biology: treasure trove or trivial pursuit", Academic Press; Gribskov, Michael, and John Devereux (1991), "Sequence analysis primer", New York, NY: M. Stockton Press; Carillo et al., 1988. SIAM J. Applied Math. 48:1073. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., 1984. Nucl Acid Res. 2:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST manual; Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990. J Mol Biol. 215:403-410). The well-known Smith Waterman algorithm may also be used to determine identity.
- The term "ligation" refers to the process of joining DNA molecules together with covalent bonds. For example, DNA ligation involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. Ligation is preferably carried out at temperature ranging from about 4 to about 37°C in the presence of a ligase enzyme. Examples of suitable ligases include Thermus thermophilus ligase, Thermus acquaticus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.
- The term "lysate" refers to a liquid or solid collection of materials following a biological unit's lysis procedure.
- The term "lysis" or "lyse" refers to the disruption of a biological unit in order to gain access to materials that are otherwise inaccessible. When the biological unit is a cell, lysis refers to breaking the cellular membrane of the cell, allowing transfer of reagents into the cell through cellular membrane holes and/or causing the cellular contents to spill out. Lysis methods are well-known to the skilled artisan, and include, but are not limited to, proteolytic lysis, chemical lysis, thermal lysis, mechanical lysis, and osmotic lysis.
- The terms "nucleic acid sequence primer" or "primer" refer to an oligonucleotide that is capable of hybridizing or annealing with a nucleic acid and serving as an initiation site for nucleotide polymerization under appropriate conditions, such as the presence of nucleoside triphosphates and an enzyme for polymerization, such as DNA or RNA polymerase or reverse transcriptase, in an appropriate buffer and at a suitable temperature.
- The term "oligonucleotide" refers to a polymer of nucleotides, generally to a single-stranded polymer of nucleotides. In some embodiments, the oligonucleotide comprises from 2 to 500 nucleotides, preferably from 10 to 150 nucleotides, preferably from 20 to 100 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically. In some embodiments, oligonucleotides may comprise ribonucleotide monomers, deoxyribonucleotide monomers, or a mix of both.
- The terms "PCR handle sequence" and "universal tag sequence" are interchangeable, and refer to a nucleic acid sequence useful for enabling amplification, preferably PCR amplification and further sequencing of nucleic acid sequences extracted or derived from the biological units. In one embodiment, the PCR handle lacks homology with the template sequence. In one embodiment, the PCR handle sequence is common for the entire sample preparation workflow.
- The term "phasing" refers to the identification of the individual complement of homologous chromosomes.
- The terms "polishing" or "blunting" refer to the elimination of incompatible 3' or 5' DNA overhangs for the promotion of blunt-end ligation. Several techniques well-known from the skilled artisan may be used for DNA end polishing. For example, terminal unpaired nucleotides may be removed from DNA ends by using an enzyme with exonuclease activity, which hydrolyzes a terminal phosphodiester bond, thereby removing the overhang one base at a time. DNA fragments with 5' overhangs may be blunted by filling in a recessed 3' terminus with DNA polymerase in the presence of dNTPs. End removal or fill-in can be accomplished using a number of enzymes, including DNA Polymerase I Large (Klenow) Fragment, T4 DNA Polymerase or Mung Bean Nuclease.
- The terms "polymerase chain reaction" or "PCR" encompass methods including, but not limited to, allele-specific PCR, asymmetric PCR, hot-start PCR, intersequence-specific PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex-PCR, nested PCR1 quantitative PCR, reverse transcription PCR and/or touchdown PCR. DNA polymerase enzymes suitable to amplify nucleic acids comprise, but are not limited to, Taq polymerase Stoffel fragment, Taq polymerase, Advantage DNA polymerase, AmpliTaq, AmpliTaq Gold, Titanium Taq polymerase, KlenTaq DNA polymerase, Platinum Taq polymerase, Accuprime Taq polymerase, Pfu polymerase, Pfu polymerase turbo, Vent polymerase, Vent exo- polymerase, Pwo polymerase, 9 Nm DNA polymerase, Therminator, Pfx DNA polymerase, Expand DNA polymerase, rTth DNA polymerase, DyNAzyme-EXT Polymerase, Klenow fragment, DNA polymerase I, T7 polymerase, SequenaseTM, Tfi polymerase, T4 DNA polymerase, Bst polymerase, Bca polymerase, BSU polymerase, phi-29 DNA polymerase and DNA polymerase Beta or modified versions thereof. In one embodiment, the DNA polymerase has a 3'-5' proofreading, i.e., exonuclease, activity. In one embodiment, the DNA polymerase has a 5'-3' proofreading, i.e., exonuclease, activity. In one embodiment, the DNA polymerase has strand displacement activity, i.e., the DNA polymerase causes the dissociation of a paired nucleic acid from its complementary strand in a direction from 5' towards 3', in conjunction with, and close to, the template-dependent nucleic acid synthesis. DNA polymerases such as E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, and HIV virus reverse transcriptase are enzymes which possess both the polymerase activity and the strand displacement activity. Agents such as helicases can be used in conjunction with inducing agents which do not possess strand displacement activity in order to produce the strand displacement effect, that is to say the displacement of a nucleic acid coupled to the synthesis of a nucleic acid of the same sequence. Likewise, proteins such as Rec A or Single Strand Binding Protein from E. coli or from another organism could be used to produce or to promote the strand displacement, in conjunction with other inducing agents (Kornberg A. & Baker T.A. (1992). Chapters 4-6. In DNA replication (2nd ed., pp. 113-225). New York: W.H. Freeman).
- The term "primer-directed extension" refers to any method known in the art wherein primers are used to initiate replication of nucleic acid sequences in the linear or logarithmic amplification of nucleic acid molecules. Primer-directed extension may be accomplished by any of several schemes known in this art including, but not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand-displacement amplification (SDA). "Primer-directed extension" can be carried out by DNA polymerase enzymes as described hereinabove.
- The term "random amplification techniques" includes without limitation, multiple displacement amplification (MDA), random PCR, random amplification of polymorphic DNA (RAPD) or multiple annealing and looping based amplification cycles (MALBAC).
- The term "receptor ligand" refers to any substance that binds to another entity, such as a receptor, from a larger complex.
- The term "reverse transcription" refers to the replication of RNA using a RNA-directed DNA polymerase (reverse transcriptase, RT) to produce complementary strands of DNA (cDNA). The reverse-transcription of RNAs may be carried out by techniques well known to the skilled artisan, using a reverse transcriptase enzyme and a mix of 4 deoxyribonucleotides triphosphate (dNTPs), namely deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and (deoxy)thymidine triphosphate (dTTP). In some embodiments, the reverse-transcription of RNAs comprises a first step of first-strand cDNA synthesis. Methods for first-strand cDNA synthesis are well-known to the skilled artisan. First-strand cDNA synthesis reactions can use a combination of sequence-specific primers, oligo(dT) primers or random primers. Examples of reverse transcriptase enzymes include, but are not limited to, M-MLV reverse transcriptase, SuperScript II (Invitrogen), SuperScript III (Invitrogen), SuperScript IV (Invitrogen), Maxima (ThermoFisher Scientific), Proto Script II (New England Biolabs), PrimeScript (ClonTech).
- The terms "single-cell epigenome profiling" or "single-cell epigenomics" refer to the analysis of the epigenome of a single-cell.
- The terms "single-cell genotyping" or "single-cell genomics" refer to the analysis of the genome of a single-cell.
- The term "single-cell haplotyping" refers to the resolution of haplotypes on a whole genome basis.
- The terms "single-cell transcriptome profiling" or "single-cell transcriptomics" refer to the analysis of the transcriptome of a single-cell.
- The term "spacer region" refers to a chemical group or an anchor moiety that is used to extend the length of an oligonucleotide. Examples of spacer include, but are not limited to, ethyleneglycol polymer, alkyl, oligonucleotides, peptides and peptidomimetics.
- The term "specific amplification techniques" includes without limitation, methods requiring temperature cycling (such as polymerase chain reaction (PCR), ligase chain reaction, transcription based amplification) and/or isothermal amplification systems (such as self-sustaining sequence replication, replicase system, helicase system, strand displacement amplification, rolling circle-based amplification and NASBA).
- The term "support" refers to a matrix upon which biological units and/or barcode units may be immobilized. The support may be rigid, solid or semi-solid.
- The terms "template" or "template sequence" refer to a nucleic acid sequence for which amplification is desired. A template can comprise DNA or RNA. In one embodiment, the template sequence is known. In one embodiment, the template sequence is not known.
- The term "template switching" refers to the ability of a reverse transcriptase to switch from an initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template (called "template switch oligonucleotide") having little or no complementarity to the 3' end of the cDNA synthesized from the initial template.
- The terms "template switch adaptor sequence" and "template switch oligonucleotide" refer to an oligonucleotide template to which a polymerase switches from an initial template (e.g., a template DNA or RNA) during a nucleic acid polymerization reaction. In this regard, the template DNA or RNA may be referred to as a "donor template" and the template switch oligonucleotide may be referred to as an "acceptor template".
- When reverse transcription occurs using a Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV reverse transcriptase), terminal nucleotidyl transferase (TdT) activity of the enzyme results in non-template-directed addition of nucleotides to the 3' end of the nascent cDNA strand. An exogenously added "template switch oligonucleotide" anneals to the C-tract by a poly(G) primer site. The reverse transcriptase then switches templates from the mRNA to the template switch oligonucleotide, adding an "adaptor sequence" or "adaptor" to the first strand cDNA (i.e. "adaptering"). Preferably, the adaptor sequence shares homology with the PCR handle.
- The term "transcriptome" refers to the entire RNA component of an individual cell. In some embodiments, the term "transcriptome" may refer specifically to the polyadenylated products of RNA polymerase II.
- The term "unique molecular identifier sequence" refers to a nucleic acid sequence useful for discriminating between amplification product duplicates after PCR amplification and further sequencing of nucleic acid sequences from the biological units.
- The term "vitamin" refers to any of a group of organic substances essential in small quantities to normal metabolism in a subject. Examples of vitamins include, but are not limited to, α-carotene, β-carotene, γ-carotene, retinol, and tretinoin (vitamin A); thiamin (vitamin B1) and analogues such as acefurtiamine, allithiamine, benfotiamine, fursultiamine, octotiamine, prosultiamine, and sulbutiamine; riboflavin (vitamin B2); niacin and nicotinic acid (vitamin B3); adenine, carnitine and choline (vitamin B4); pantothenic acid, dexpanthenol, and pantethine (vitamin B5); pyridoxine, pyridoxal phosphate, pyridoxamine, and pyritinol (vitamin B6); biotin (vitamin B7); adenosine monophosphate (AMP) and inositol (vitamin B8); folic acid, dihydrofolic acid, folinic acid, and levomefolic acid (vitamin B9); 4-aminobenzoic acid (pABA) (vitamin B10); pteryl-hepta-glutamic acid (PHGA) (vitamin B11); adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin (vitamin B12); orotic acid (vitamin B13); pangamic acid (vitamin B15); dimethylglycine (DMG) (vitamin B16); amygdalin (vitamin B17); L-carnitine (vitamin B20); ascorbic acid, and dehydroascorbic acid (vitamin C); ergosterol, and ergocalciferol (vitamin D2); 7-dehydrocholesterol, previtamin D3, cholecalciferol, 25-hydroxycholecalciferol, calcitriol, and calcitroic acid (vitamin D3); dihydroergocalciferol (vitamin D4); alfacalcidol, dihydrotachysterol, calcipotriol, tacalcitol, and paricalcitol (vitamin D5); α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, and tocofersolan (vitamin E); phylloquinone (vitamin K1); menaquinones (vitamin K2); menadione (vitamin K3); menadiol (vitamin K4); and derivatives thereof.
- The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".
- The present invention relates to methods for trapping and barcoding discrete biological units in a hydrogel. In one embodiment, a plurality of biological units is bound on a support. In one embodiment, a plurality of barcode units is bound on a support.
- In one embodiment, the method comprises contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes. In one embodiment, the method further comprises contacting the biological unit/barcode unit complexes with a hydrogel solution. In one embodiment, the method further comprises polymerizing the hydrogel solution to embed the biological unit/barcode unit complex in a hydrogel matrix. In one embodiment, the method further comprises barcoding the biological unit's nucleic acid within each biological unit/barcode unit complex in the hydrogel matrix.
- In one embodiment, the biological units and barcode units unbind after hydrogel polymerization, i.e., the biological unit/barcode unit complexes' binding chemistry is degraded. Techniques to break down complexes are well-known to the skilled artisan.
- In one embodiment, biochemistry and molecular biology assays can be performed on biological units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on discrete biological units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on barcode units trapped in a hydrogel according to the present invention. In one embodiment, biochemistry and molecular biology assays can be performed on discrete barcode units trapped in a hydrogel according to the present invention. In one embodiment, the hydrogel can be depolymerized to allow for certain biochemistry and molecular biology assays in solution and/or in bulk.
- Examples of biochemistry and molecular biology assays include, but are not limited to, cell lysis, PCR, reverse transcription, nucleic acid hydrolyzing, decapping (i.e., hydrolysis of a 5' cap structure), transcriptome profiling (or transcriptomics), genotyping (or genomics), epigenome profiling (or epigenomics), phasing, and haplotyping.
- Several aspects of the methods according to the present invention are described herein with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. One having ordinary skill in the relevant art, however, will readily recognize that the features described herein can be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.
- Hydrogels can be classified into physical and chemical hydrogels based on their cross-linking mechanism.
- In one embodiment, hydrogels are prepared from at least one natural polymer. In one embodiment, hydrogels are prepared from at least one synthetic polymer. In one embodiment, hydrogels are prepared from at least one natural/synthetic hybrid polymer. In one embodiment, hydrogels are prepared from at least one natural polymer and at least one synthetic polymer.
- In one embodiment, the hydrogels used in the present invention are physical hydrogels.
- Physical hydrogel crosslinks include, but are not limited to, entangled chains, hydrogen bonding, hydrophobic interaction and crystallite formation. Physical hydrogel can be synthesized by ionic interaction, crystallization, stereocomplex formation, hydrophobized polysaccharides, protein interaction and hydrogen bond.
- In one embodiment, physical hydrogels are permanent. In one embodiment, physical hydrogels are reversible. In one embodiment, the hydrogels used in the present invention are chemical hydrogels.
- Chemical hydrogels crosslinks include, but are not limited to, covalent bounds. Chemical hydrogels can be synthesized by chain growth polymerization, addition and condensation polymerization and gamma and electron beam polymerization.
- In one embodiment, chemical hydrogels are formed by polymerization of end-functionalized macromers.
- In one embodiment, chemical hydrogels are permanent. In one embodiment, chemical hydrogels are reversible. In one embodiment, hydrogels are polysaccharide hydrogels.
-
- In one embodiment, polymerized polysaccharide hydrogels are formed by covalent crosslinking, ionic crosslinking, chemical conjugation, esterification and/or polymerization.
- In one embodiment, polysaccharide hydrogel is alginate and polymerized alginate is formed by ionic crosslinking in presence of a divalent cation, such as calcium.
- In one embodiment, hydrogels are protein-based hydrogels.
- Proteins include, but are not limited to, collagen, fibrin, gelatin, laminin.
- In one embodiment, polymerized protein-based hydrogels are formed by thermal gelation. In one embodiment, protein-based hydrogels are crosslinked using a crosslinker.
- Protein-based hydrogels' crosslinkers include, but are not limited to, carbodiimide, cyanamide, dialdehyde starch, diimide, diisocyanate, dimethyl adipimidate, epoxy compounds, ethylaldehyde, formaldehyde, glutaraldehyde, glyceraldehyde, hexamethylenediamine, terephthalaldehyde and mixture thereof.
- In one embodiment, hydrogels are polysaccharide hydrogels combined with proteins as described here above.
- In one embodiment, hydrogels are nonbiodegradable synthetic hydrogels.
- Nonbiodegradable polymers include, but are not limited to, vinylated monomers and vinylated macromers, in particular, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, acrylamide, acrylic acid, N-isopropylacrylamide, poly N-isopropylacrylamide, methoxypolyethylene glycol monoacrylate.
- In one embodiment, nonbiodegradable molecule polymerization requires at least one crosslinker. In one embodiment, nonbiodegradable synthetic hydrogels are formed by copolymerization of a nonbiodegradable molecules and a crosslinker.
- Nonbiodegradable synthetic hydrogels' crosslinkers include, but are not limited to, N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate.
- In one embodiment, nonbiodegradable molecule polymerization further requires at least one initiator, such as, e.g., persulfate ions (ammonium persulfate, potassium persulfate and the like), ammonium cerium (IV) nitrate, tetramethylethylenediamine (TEMED).
- In one embodiment, the hydrogel can be depolymerized. By "depolymerization" is meant a reaction during which the hydrogel returns in solution. As will clearly appear to the skilled person, this does not necessarily require extensive depolymerization and/or extensive breakage of crosslinks. The extent of depolymerization and/or breakage of crosslinks required to achieve gel-to-sol transition will depend on the nature of the hydrogel and can be readily determined by common methods. In one embodiment, depolymerization of the hydrogel is chemical. In one embodiment, depolymerization of the hydrogel is thermal. In one embodiment, depolymerization of the hydrogel is enzymatic.
- In one embodiment, depolymerization of the hydrogel can be achieved by divalent cation removal. Examples of hydrogels which can be depolymerized by divalent cation removal include, but are not limited to, alginate. In one embodiment, depolymerization of the hydrogel can be achieved by addition of reducing agent. Examples of reducing agents include, but are not limited to, phosphines (e.g., tris(2-carboxyethyl)phosphine (TCEP)) and dithiothreitol (DTT). Examples of hydrogels which can be depolymerized by addition of reducing agent include, but are not limited to, hydrogels copolymerized with a crosslinker such as nonbiodegradable synthetic hydrogels.
- In one embodiment, depolymerization of the hydrogel can be achieved by thermal melting, i.e., melting upon increase of the temperature.
- In one embodiment, the hydrogel used in the present invention is thermosensitive.
- By "thermosensitive" is meant a hydrogel which, after being formed, depolymerizes if raised above the melting point of the at least one polymer, and reforms if cooled to room temperature or below its melting point. In one embodiment, the hydrogel used in the present invention is thermoreversible.
- By "thermoreversible" is meant a hydrogel which, after being formed, depolymerizes if raised above the melting point of the at least one polymer and does not reform, even when cooled to room temperature or below its melting point.
- In one embodiment, the melting point of the at least one polymer of the hydrogel is between about 20°C and about 200°C, preferably between about 25°C and about 100°C.
- In one embodiment, the hydrogel has a pore size sufficiently small to trap a biological unit, a barcode unit and/or an analyte extracted or derived from a biological unit. In one embodiment, the hydrogel has a pore size sufficiently large to allow diffusion of biochemistry and molecular biology reagents.
- In one embodiment, the hydrogel has a pore size ranging between about 1 nm and 1 µm, preferably between about 10 nm and 500 nm, more preferably between 25 nm and 250 nm.
- In one embodiment, the hydrogel matrix is accessible to biochemistry and molecular biology reagents. In one embodiment, the hydrogel matrix has at least one surface accessible to biochemistry and molecular biology reagents. In one embodiment, the at least one surface accessible to biochemistry and molecular biology reagents is naturally occurring. In one embodiment, the at least one surface accessible to biochemistry and molecular biology reagents is shaped before, during and/or after hydrogel polymerization.
- In one embodiment, the composition, shape, form, and modifications of the barcode unit can be selected from a range of options depending on the application.
- Exemplary materials that can be used as a barcode unit in the present invention include, but are not limited to, acrylics, carbon (e.g., graphite, carbon-fiber), cellulose (e.g., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g., agarose, SEPHAROSE™ or alginate), gels, glass (e.g., modified or functionalized glass), gold (e.g., atomically smooth Au(111)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides (e.g., SiO2, TiO2, stainless steel), metalloids, metals (e.g., atomically smooth Au(111)), mica, molybdenum sulfides, nanomaterials (e.g., highly oriented pyrolitic graphite (HOPG) nanosheets), nitrocellulose, NYLON™, optical fiber bundles, organic polymers, paper, plastics, polacryloylmorpholide, poly(4-methylbutene), polyethylene terephthalate), poly(vinyl butyrate), polybutylene, polydimethylsiloxane (PDMS), polyethylene, polyformaldehyde, polymethacrylate, polypropylene, polysaccharides, polystyrene, polyurethanes, polyvinylidene difluoride (PVDF), quartz, rayon, resins, rubbers, semiconductor material, silica, silicon (e.g., surface-oxidized silicon), sulfide, and TEFLON™.
- In one embodiment, the barcode unit is composed of a single material. In another embodiment, the barcode unit is composed of a mixture of several different materials.
- In one embodiment, the barcode units used in the present invention can be simple square grids, checkerboard grids, hexagonal arrays and the like. Suitable barcode units also include, but are not limited to, beads, slides, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, microtiter plates such as 768-well, 384-well, 96-well, 48-well, 24-well, 12-well, 8-well, 6-well, 4-well, 1-well and the like. In various embodiments, the barcode unit may be biological, non-biological, organic, inorganic, or any combination thereof.
- Accordingly, a single barcode unit in a plurality of barcode units may be a minimal, indivisible part of said plurality of barcode units. A single barcode unit in a plurality of barcode units may be, e.g., a single square on a grid, a single bead in a population of beads, a single well in a microtiter plate, etc. Alternatively, a single barcode unit in a plurality of barcode units may be a minimal part of said plurality of barcode units, wherein a single binding event between a biological unit and a barcode unit occurs at the molecular level. Alternatively, a single barcode unit in a plurality of barcode units may be a part of said plurality of barcode units ranging from about 1 µm2 to about 1 mm2, preferably from about 1 µm2 to about 100 µm2, more preferably from about 1 µm2 to about 50 µm2. In one embodiment, this size range is chosen for manufacturability. In one embodiment, this size range is chosen to ensure the formation of biological unit/barcode unit complexes with a 1:1 ratio. The surface of the barcode unit can be modified according to methods known to the skilled artisan, to promote trapping or immobilization of biological units thereon.
- In one embodiment, the barcode unit comprises reactive groups on its surface, such as carboxyl, amino, hydroxyl, epoxy, and the like.
- In one embodiment, the barcode unit can have functional modifications, such as functional groups attached to its surface.
- In one embodiment, the barcode unit used in the present invention is barcoded.
- In one embodiment, each single barcode unit in a plurality of barcode units comprises a unique barcode. In one embodiment, each single barcode unit in a plurality of barcode units comprises clonal copies of a unique barcode.
- In one embodiment, the barcode unit comprises at least one means involved with binding at least one biological unit.
- In a preferred embodiment, the barcode unit is a bead.
- The implementation of methods according to the present invention may rely on the downstream identification of each discrete biological unit and/or of the reactional analytes bound to each barcode unit. Therefore, it may be desirable to add at least one identifier or barcode to the barcode unit, in order to convey information about the source or origin of the biological unit and/or of an analyte within a sample, such as for example, a nucleic acid sequence extracted or derived from a discrete biological unit.
- In one embodiment, the barcode unit is barcoded. In one embodiment, each single barcode unit in a plurality of barcode units comprises a unique barcode. In one embodiment, each single barcode unit in a plurality of barcode units comprises clonal copies of a unique barcode.
- Barcodes may be of a variety of different formats, including labels, tags, probes, and the like.
- In one embodiment, the barcode unit is optically barcoded. In one embodiment, the barcode unit is non-optically barcoded. In one embodiment, the barcode unit is optically and non-optically barcoded.
- Optical barcodes include, but are not limited to, chromophores, fluorophores, quantum dots, styrene monomers, and combination thereof, which can be identified, e.g., by their spectrum such as Raman spectrum or electromagnetic spectrum; and/or by their intensity of color.
- Non-optical barcodes include, but are not limited to, biomolecular sequences such as DNA, RNA and/or protein sequences, which can be identified, e.g., by sequencing.
- In one embodiment, the number of unique barcodes used in the present invention ranges from about 2 to about 1012.
- In one embodiment, the number of clonal copies of each unique barcode comprised in each single barcode unit in a plurality of barcode units ranges from about 2 to about 1012.
- In one embodiment, the barcode unit according to the present invention comprises non-optical barcodes. In one embodiment, the barcode unit according to the present invention comprises nucleic acid barcodes. In one embodiment, the nucleic acid barcode is single stranded. In one embodiment, the nucleic acid barcode is double stranded. In one embodiment, the nucleic acid barcode is single and/or double stranded. In one embodiment, the barcode unit according to the present invention comprises DNA barcodes. In one embodiment, the barcode unit according to the present invention comprises RNA barcodes. In one embodiment, the barcode unit according to the present invention comprises a mixture of DNA and RNA barcodes.
- In one embodiment, the nucleic acid barcode according to the present invention comprises from 5 to 20 nucleotides, preferably from 8 to 16 nucleotides.
- In one embodiment, the barcode unit comprises a plurality of unique nucleic acid sequences, i.e., clonal copies of a unique barcode.
- In one embodiment, said unique nucleic acid sequences are degenerate sequences. In one embodiment, said unique nucleic acid sequences are based on combinatorial chemistry.
- Techniques to covalently attach barcodes on a support, preferably on a barcode unit, are well known to the skilled artisan, and include without limitation, replication of bound primers in a combinatorial fashion, ligation of adaptors in a combinatorial fashion, and chemical addition of nucleotides in a combinatorial fashion.
- In one embodiment, said unique nucleic acid sequences are amplified on the barcode unit such that each single barcode unit in a plurality of barcode units is coated with clonal copies of a starting nucleic acid sequence.
- In one embodiment, the covalent attachment of nucleic acid barcodes to the barcode unit is carried out directly during synthesis of the barcodes. In one embodiment, the covalent attachment of nucleic acid barcodes to the barcode unit is carried out after synthesis of the barcode.
- Techniques to covalently attach nucleic acid barcodes onto a barcode unit are well known to the skilled artisan. In one embodiment, barcoding of the biological unit's nucleic acid is achieved by primer template annealing of the barcode to the biological unit's nucleic acid. In one embodiment, barcoding of the biological unit's nucleic acid is achieved by primer-directed extension of the barcode to the biological unit's nucleic acid. In one embodiment, barcoding of the biological unit's nucleic acid is achieved by ligation of the barcode to the biological unit's nucleic acid.
- The implementation of the methods according to the present invention may rely on the immobilization, replication, extension and/or amplification of nucleic acid sequences of or from the biological units. Therefore, it may be desirable to add at least one nucleic acid sequence primer to the barcode unit, preferably at least one nucleic acid sequence primer to each single barcode unit in a plurality of barcode units, in order to immobilize, replicate, extend and/or amplify genetic information of or from the biological units.
- In one embodiment, the nucleic acid sequence primer is single-stranded. In one embodiment, the nucleic acid sequence primer is double-stranded. In one embodiment, the nucleic acid sequence primer is single-stranded and/or double-stranded.
- In one embodiment, the nucleic acid sequence primer is a degenerate (i.e., random) nucleic acid sequence primer. In one embodiment, the nucleic acid sequence primer is specific to a nucleic acid sequence of interest. In one embodiment, the nucleic acid sequence primer can prime at multiple locations of the nucleic acid sequences of or from the biological units. In one embodiment, the nucleic acid sequences of or from the biological units comprise multiple priming sites.
- In one embodiment, the nucleic acid sequence primer comprises a poly-dT sequence. In one embodiment, the nucleic acid sequence primer comprises a poly-dU sequence. Accordingly, the nucleic acid sequence primer is specific to a poly-A sequence. Poly-A sequences may be found, e.g., on the 3' end of mRNAs, within the poly-A tail.
- In one embodiment, the nucleic acid sequence primer comprises the sequence (dT)nVN, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e., A, C or G), and N represents any nucleotide (i.e., A, T/U, C or G). In one embodiment, the nucleic acid sequence primer comprises the sequence (dU)nVN, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e., A, C or G), and N represents any nucleotide (i.e., A, T/U, C or G). Accordingly, the nucleic acid sequence primer is specific to a (A)nBN sequence, wherein n ranges from 5 to 50, B represents any nucleotide but A (i.e., T/U, C or G), and N represents any nucleotide (i.e., A, T/U, C or G). (A)nBN sequences may be found, e.g., on the 3' end of mRNAs, overlapping between the poly-A tail and the 3' UTR or CDS.
- In one embodiment, the nucleic acid sequence primer comprises a poly-I sequence. Accordingly, the nucleic acid sequence primer is non-specific and can prime to any nucleic acid sequence of or from the biological units. In one embodiment, the nucleic acid sequence primer comprises from 5 to 50 nucleotides, preferably from 5 to 30 nucleotides.
- In one embodiment, the covalent attachment of nucleic acid sequence primers to the barcode unit is carried out directly during synthesis of the nucleic acid sequence primers. In one embodiment, the covalent attachment of nucleic acid sequence primers to the barcode unit is carried out after synthesis of the nucleic acid sequence primers.
- In one embodiment, the barcode unit comprises at least one oligonucleotide.
- In one embodiment, the at least one oligonucleotide is a DNA oligonucleotide. In one embodiment, the at least one oligonucleotide is a RNA oligonucleotide. In one embodiment, the at least one oligonucleotide is a DNA/RNA hybrid oligonucleotide.
- In one embodiment, the at least one oligonucleotide is single-stranded. In one embodiment, the at least one oligonucleotide is double-stranded. In one embodiment, the at least one oligonucleotide is single-stranded and/or double-stranded.
- In one embodiment, the at least one oligonucleotide comprises at least one nucleic acid barcode and at least one nucleic acid sequence primer. In one embodiment, the at least one oligonucleotide comprises from 5' to 3' at least one nucleic acid barcode and at least one nucleic acid sequence primer. In one embodiment, the at least one oligonucleotide comprises from 5' to 3' at least one nucleic acid sequence primer and at least one nucleic acid barcode. In one embodiment, the nucleic acid barcodes are identical across all oligonucleotides on the surface of a given barcode unit. In one embodiment, the nucleic acid barcodes are different across oligonucleotides on the surface of one barcode unit with respect to another barcode unit. In one embodiment, the nucleic acid sequence primer is identical across all oligonucleotides on the surface of a given barcode unit. In one embodiment, the nucleic acid sequence primer is different across all oligonucleotides on the surface of a given barcode unit. In one embodiment, the nucleic acid sequence primer is identical across all oligonucleotides and barcode units. In one embodiment, the nucleic acid barcode comprises from 5 to 20 nucleotides, preferably from 8 to 16 nucleotides. In one embodiment, the nucleic acid sequence primer comprises from 5 to 50 nucleotides, preferably from 5 to 30 nucleotides.
- In one embodiment, the at least one oligonucleotide further comprises a PCR handle sequence. In one embodiment, the PCR handle sequence is identical across all oligonucleotides and barcodes units. In one embodiment, the PCR handle sequence comprises from 10 to 30 nucleotides, preferably from 15 to 25 nucleotides.
- In one embodiment, the at least one oligonucleotide further comprises a unique molecular identifier sequence. In one embodiment, the unique molecular identifier sequence is different across all oligonucleotides on the surface of a given barcode unit. In one embodiment, the unique molecular identifier sequence comprises from 10 to 30 nucleotides, preferably from 15 to 25 nucleotides.
- In one embodiment, the at least one oligonucleotide further comprises a spacer region.
- In one embodiment, the at least one oligonucleotide comprises, from 5' to 3' (i.e., from proximal to distal with regard to the surface of the barcode unit):
- optionally, a spacer region;
- optionally, a PCR handle sequence;
- a nucleic acid barcode;
- optionally, a unique molecular identifier sequence; and
- a nucleic acid sequence primer.
- In one embodiment, the at least one oligonucleotide comprises, from 3' to 5' (i.e., from distal to proximal with regard to the surface of the barcode unit):
- optionally, a spacer region;
- optionally, a PCR handle sequence;
- a nucleic acid barcode;
- optionally, a unique molecular identifier sequence; and
- a nucleic acid sequence primer.
- In one embodiment, the covalent attachment of nucleic acid oligonucleotides to the barcode unit is carried out directly during synthesis of the nucleic acid oligonucleotides. In one embodiment, the covalent attachment of nucleic acid oligonucleotides to the barcode unit is carried out after synthesis of the nucleic acid oligonucleotides.
- Techniques to covalently attach and/or to synthesize nucleic acid oligonucleotides onto a barcode unit such as glass or plastic tubes or beads, nitrocellulose or nylon filters, microtiter wells, agarose bead gels and magnetic particles are well known to the skilled artisan. These include, but are not limited to, UV irradiation, biotin-avidin/streptavidin and covalent chemical attachment (Macosko et al., 2015. Cell. 161:1202-1214 ; Fan et al., 2015. Science. 347(6222):1258367; Beaucage, S.L. (1993), In Protocols for oligonucleotides and analogs-Synthesis and properties. Totowa, NJ: Humana Press, 20:33-61; Conner et al., 1983. Proc Natl Acad Sci. USA. 80:278-282; Lockley et al., 1997. Nucleic Acids Res. 25:1313-1314; Joos et al., 1997. Anal Biochem. 247:96-101; Cohen et al., 1997. Nucl Acid Res. 25:911-912; Yang et al., 1998. Chem Lett. 3:257-258; Maskos et al., 1992. Nucl Acid Res. 20:1679-1684; Chrisey et al., 1996. Nucl Acid Res. 24:3131-3039; Chrisey et al., 1996. Nucl Acid Res. 24:3040-3047; Marble et al., 1995. Biotechnol Prog. 11:393-396; Liu et al., 1997. Promega Notes Mag. 64:21-25; Weiler et al., 1996. Anal Biochem. 243:218-227; Beattie et al., 1995. Mol Biotechnol. 4:213-225; Rasmussen et al., 1991. Anal Biochem. 198:138-142; Timofeev et al., 1996. Nucl Acid Res. 24:3142-3148; Yershov et al., 1997. Anal Biochem. 250:203-211; DeAngelis et al., 1995. Nucl Acid Res. 23:4742-4743; Haukanes et al., 1993. Biotechnology. 11:60-63).
- The implementation of the methods according to the present invention may rely on the binding and/or the immobilization of a biological unit on the barcode unit. Therefore, it may be desirable to add at least one means for binding a biological unit to the barcode unit, in order to trap discrete biological units.
- In one embodiment, the binding and/or the immobilization of a biological unit to the barcode unit is aspecific. In one embodiment, the binding and/or the immobilization of a biological unit to the barcode unit is specific. In one embodiment, the binding and/or the immobilization of a biological unit on the barcode unit requires the presence of at least one means for binding a barcode unit on the biological unit.
- Means for binding a biological unit and/or means for binding a barcode unit comprise, but is not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, a hydrophobic group.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a protein and/or at least a peptide. Examples of proteins or peptides include, but are not limited to, antibodies (e.g., IgA, IgD, IgE, IgG, and IgM) and fragments thereof, including, but not limited to, Fab fragments, F(ab')2 fragments, scFv fragments, diabodies, triabodies, scFv-Fc fragments, minibodies; protein A, protein G, avidin, streptavidin, receptors and fragments thereof, and ligands and fragments thereof.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a nucleic acid. Examples of nucleic acids include, but are not limited to, DNA, RNA and artificial nucleic acids, such as nucleic acids comprising inosine, xanthosine, wybutosine, and/or analogs thereof.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a carbohydrate. Examples of carbohydrates include, but are not limited to, monosaccharides, disaccharides and polysaccharides.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a vitamin.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a coenzyme.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a receptor ligand.
- In one embodiment, the means for binding a biological unit and/or the means for binding a barcode unit comprise at least a hydrophobic group. Examples of hydrophobic groups include, but are not limited to, alkyl groups having from about 2 to about 8 carbon atoms, such as an ethyl, propyl, butyl, pentyl, heptyl, or octyl and isomeric forms thereof; or aryl groups such as phenyl, benzyl or naphthyl.
- Techniques for coating a barcode unit with a means for binding a biological unit are well-known to the skilled artisan.
- In one embodiment, the coating may be an all-over coating, i.e., completely covering the barcode unit, or may be a partial coating, i.e., covering only parts of the barcode unit.
- In one embodiment, coating of a barcode unit with a means for binding a biological unit requires functionalization of the barcode unit. Examples of functionalized barcode units include, but are not limited to, amino-functionalized barcode units, carboxyl-functionalized barcode units, hydroxyl-functionalized barcode units and epoxy-functionalized barcode units. Techniques to functionalize a barcode unit are well-known in the art and include, but are not limited to, organosilane crosslinking, such as methoxysilane, ethoxysilane and acetoxysilane derivatives.
- Examples of techniques for coating a barcode unit with a means for binding a biological unit include, but are not limited to, adsorption and covalent attachment. Covalent attachment may be performed on functionalized barcode units, using coupling agents such as carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfo-NHS, dimethylaminopropyl (DEAP), glutaraldehyde, aldehyde, sodium cyanoborohydride (NaCNBH3), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), dithiothreitol (DTT), and/or cyanogen bromide (BrNC). In one embodiment, the at least one means for binding a barcode unit is naturally present on and/or in the biological unit. In one embodiment, the at least one means for binding a barcode unit is not naturally present on and/or in the biological unit.
- In one embodiment, the biological unit is incubated with at least one antibody prior to the binding and/or the immobilization on the barcode unit. In one embodiment, the at least one antibody is specific towards the biological unit. In one embodiment, the at least one antibody is functionalized. Examples of functionalization include, but are not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, and a hydrophobic group. In one embodiment, the antibody is biotinylated, i.e., is functionalized with a biotin moiety. The implementation of the methods according to the present invention may rely on the binding and/or the immobilization of a single biological unit on a single barcode unit. Therefore, it may be desirable to prevent more than one biological unit from binding to each barcode unit; or alternatively, to prevent more than one barcode unit from binding to each biological unit.
- Depending on parameters such as the concentration and/or the size of both the biological units and the barcode units, more than one biological unit can bind to a single barcode unit, and vice versa. Consequently, the methods according to the present invention provides means for ensuring, selecting and/or purifying biological unit/barcode unit complexes with a 1:1 ratio. The methods according to the present invention also provides means for forming biological unit/barcode unit complexes with a 1:1 ratio.
- In one embodiment, the methods of the present invention comprise a step of selection and/or purification of biological unit/barcode unit complexes with a 1:1 ratio. According to one embodiment, a plurality of biological units may be contacted with a plurality of barcode units to form biological unit/barcode unit complexes, which may be further selected and/or purified.
- Techniques to select and/or purify complexes are well known to the skilled artisan, and include, but are not limited to, size exclusion chromatography techniques, density gradient techniques, and/or filtration techniques. In one embodiment, the methods of the present invention comprise a means for forming biological unit/barcode unit complexes with a 1:1 ratio.
- In one embodiment, the biological units are bound to a support. In one embodiment, the barcode units are bound to a support.
- Binding a plurality of biological units to a support prior to contacting them with a plurality of barcode units creates hindrance and allows the support to act as an impediment, preventing multiple binding of barcode units to a single biological unit. It may thus be desirable to use larger barcode units with respect to the biological units. Additionally, a limiting concentration of barcode units with respect to the biological units may be used to ensure the binding of at most one barcode unit per biological unit.
- Alternatively, binding a plurality of barcode units to a support prior to contacting them with a plurality of biological units creates hindrance and allows the support to act as an impediment, preventing multiple binding of biological units to a single barcode unit. It may thus be desirable to use smaller barcode units with respect to the biological units. Additionally, a limiting concentration of biological units with respect to the barcode units may be used to ensure the binding of at most one biological unit per barcode unit.
- In one embodiment, the composition, shape, form, and modifications of the support can be selected from a range of options depending on the application.
- Exemplary materials that can be used as a support in the present invention include, but are not limited to, acrylics, carbon (e.g., graphite, carbon-fiber), cellulose (e.g., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g., agarose, SEPHAROSE™ or alginate), gels, glass (e.g., modified or functionalized glass), gold (e.g., atomically smooth Au(111)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides (e.g., SiO2, TiO2, stainless steel), metalloids, metals (e.g., atomically smooth Au(111)), mica, molybdenum sulfides, nanomaterials (e.g., highly oriented pyrolitic graphite (HOPG) nanosheets), nitrocellulose, NYLON™, optical fiber bundles, organic polymers, paper, plastics, polacryloylmorpholide, poly(4-methylbutene), polyethylene terephthalate), poly(vinyl butyrate), polybutylene, polydimethylsiloxane (PDMS), polyethylene, polyformaldehyde, polymethacrylate, polypropylene, polysaccharides, polystyrene, polyurethanes, polyvinylidene difluoride (PVDF), quartz, rayon, resins, rubbers, semiconductor material, silica, silicon (e.g., surface-oxidized silicon), sulfide, and TEFLON™.
- In one embodiment, the support is composed of a single material. In another embodiment, the support is composed of a mixture of several different materials.
- In one embodiment, the support used in the present invention may be tubes, beads, slides, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, microtiter plates such as 768-well, 384-well, 96-well, 48-well, 24-well, 12-well, 8-well, 6-well, 4-well, 1-well, square grids, checkerboard grids, hexagonal arrays and the like. In various embodiments, the support may be biological, non-biological, organic, inorganic, or any combination thereof.
- The surface of the support can be modified according to methods known to the skilled artisan, to promote trapping or immobilization of biological units and/or barcode units thereon.
- In one embodiment, the trapping or immobilization of a biological unit and/or of a barcode unit to the support is aspecific.
- In one embodiment, biological units and/or barcode units are trapped or immobilized in a layer of hydrogel that coats the support.
- In one embodiment, the trapping or immobilization of a biological unit and/or of a barcode unit to the support is specific.
- In one embodiment, the support comprises reactive groups on its surface, such as carboxyl, amino, hydroxyl, epoxy, and the like. In one embodiment, the support can have functional modifications, such as functional groups attached to its surface. In one embodiment, the support comprises at least one means involved with binding at least one biological unit and/or at least one barcode unit.
- Means for binding a biological unit and/or a barcode unit comprise, but are not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, and a hydrophobic group, as described hereinabove.
- Techniques for coating a support with a means for binding a biological unit and/or a barcode unit are well-known to the skilled artisan.
- In one embodiment, the coating may be an all-over coating, i.e., completely covering the support, or may be a partial coating, i.e., covering only parts of the support.
- In one embodiment, coating of a support with a means for binding a biological unit and/or a barcode unit requires functionalization of the support. Examples of functionalized supports include, but are not limited to, amino-functionalized supports, carboxyl-functionalized supports, hydroxyl-functionalized supports, and epoxy-functionalized supports. Techniques to functionalize a support are well-known in the art and include, but are not limited to, organosilane crosslinking, such as methoxysilane, ethoxysilane and acetoxysilane derivatives.
- Examples of techniques for coating a support with a means for binding a biological unit and/or a barcode unit include, but are not limited to, adsorption and covalent attachment. Covalent attachment may be performed on functionalized supports, using coupling agents such as carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfo-NHS, dimethylaminopropyl (DEAP), glutaraldehyde, aldehyde, sodium cyanoborohydride (NaCNBH3), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), dithiothreitol (DTT), and/or cyanogen bromide (BrNC). In one embodiment, the method for trapping discrete biological units in a hydrogel, according to the present invention, comprises the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed said biological unit/barcode unit complexes in a hydrogel matrix, and
- d) barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- In one embodiment, each barcode unit comprises at least a means involved with binding a biological unit as defined hereinabove. In one embodiment, each biological unit comprises at least a means involved in binding the barcode unit as defined hereinabove.
- In one embodiment, each barcode unit comprises a unique barcode as defined hereinabove. In one embodiment, each barcode unit comprises clonal copies of a unique barcode.
- In one embodiment, each barcode unit comprises at least one nucleic acid sequence primer as defined hereinabove.
- In one embodiment, each barcode unit comprises a nucleic acid oligonucleotide as defined hereinabove.
- In one embodiment, the plurality of biological units is bound to a support as defined hereinabove. In one embodiment, the plurality of barcode units is bound to a support as defined hereinabove.
- In one embodiment, the methods according to the present invention may comprise a step of selection and/or sorting of the biological units. Selection and/or sorting of biological units may be based on the expression of a given surface molecule such as a protein or a carbohydrate, or on specific light scattering and fluorescence characteristics of each biological unit. Selection and/or sorting of biological units may also be bases on their size. Methods to select and/or sort biological units are well-known to the skilled artisan, and comprise, but are not limited to, fluorescent activated cell sorting (FACS), fluorescence in situ hybridization-flow cytometry (FISH-FC), IsoRaft array, DEPArray lab-on-a-chip technology, magnetic cell sorting, immunoprecipitation, filtration and the like.
- In one embodiment, the methods according to the present invention may comprise a step of lysis of the biological units.
- In one embodiment, the methods according to the present invention may comprise a step of reverse transcription of the biological units' RNA content, preferably of the biological units' mRNA content.
- In one embodiment, biochemistry and molecular biology assays can be carried out before, during or after the step of barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- In one embodiment, the methods according to the present invention may comprise a step of pre-amplification of the biological units' nucleic acids, such as DNA, RNA or cDNA. In one embodiment, the methods according to the present invention may comprise a step of pre-amplification of the biological units' nucleic acids, such as DNA, RNA or cDNA, before the step of barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- In one embodiment, the methods according to the present invention may comprise a step of purifying templates for biochemistry and molecular biology assays. Endogenous or exogenous proteins and complexes bound to nucleic acid templates or membranes encapsulating nucleic acid templates can be removed from the hydrogel after biological unit/barcode unit complex trapping. Techniques for nucleic acid purification are well known to the skilled artisan and include, without limitation, the use of proteinase K and/or detergents such as SDS, sarkosyl, NP-40, and the like.
- In one embodiment, the methods according to the present invention may comprise a step of cleaning amplified nucleic acids. Prior to preparing a nucleic acid library for sequencing, it can be desirable to remove single-stranded primers and reaction products such as enzymes. Techniques for nucleic acid clean-up are well known to the skilled artisan, and include without limitation, the use of single-strand-specific nucleases and/or the use of phosphatases to dephosphorylate phosphorylated ends of nucleic acids. Examples of single-strand-specific nucleases include, but are not limited to, exonuclease I, mung bean nuclease, nuclease Bh1, nuclease P1, nuclease S1, BAL 31 nuclease. Examples of phosphatases include, but are not limited to, alkaline phosphatase such as shrimp alkaline phosphatase.
- In one embodiment, the methods according to the present invention may comprise a step of sizing the amplified nucleic acids. Short-read sequencers, such as Illumina or Ion Torrent, operate best when fed DNA libraries that contain fragments of similar sizes, according to the manufacturer's recommendations. When libraries are not properly size-selected, these sequencers can become less efficient. Techniques for DNA size selection are well known to the skilled artisan, including, but not limited to, nucleic acid gel electrophoresis, bead-based protocols, pulsed-field gel electrophoresis (PFGE), automated size selection.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library fragmentation.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library enzymatic fragmentation.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library mechanical fragmentation.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library polishing.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library A-tailing.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acids and/or cDNA library ligation.
- In one embodiment, the methods according to the present invention may comprise a step of tagmentation. Techniques for tagmenting nucleic acids and/or cDNA library are well known to the skilled artisan.
- In one embodiment, the methods according to the present invention may comprise a step of nucleic acid sequencing. In one embodiment, the sequencing of nucleic acids may be carried out by next generation sequencing (NGS). Methods for NGS of nucleic acid libraries are known to the skilled artisan, and comprise, but are not limited to, paired-end sequencing, sequencing by synthesis, and single-read sequencing.
- In one embodiment, the methods according to the present invention comprise contacting the hydrogel matrix with biochemistry and molecular biology reagents, useful to carry out the method. In one embodiment, the hydrogel matrix is porous enough to allow diffusion of biochemistry and molecular biology reagents, without allowing diffusion of the barcode unit, biological unit and/or analytes, such as for example, nucleic acids extracted or derived from a discrete biological unit. In one embodiment, subsequent steps can be performed by exchanging and/or washing biochemistry and molecular biology reagents in contact with the hydrogel matrix. Biochemistry and molecular biology reagents are well-known to the skilled artisan, and encompass all reagents known to perform biochemistry and molecular biology assays, such as solutions (buffer solutions, wash solutions, and the like), detergents, enzymes, nucleic acid primers, and the like.
- In one embodiment, diffusion of biochemistry and molecular biology reagents is a passive diffusion. Passive diffusion includes, but is not limited to, osmosis and diffusiophoresis.
- In one embodiment, diffusion of biochemistry and molecular biology reagents is an active diffusion. Techniques for active diffusion in a hydrogel are well-known to the skilled artisan, and include, but are not limited to, the use of pumps, electroosmosis and electrophoresis.
- In one embodiment, subsequent steps are performed by exchanging the majority reagent in contact with the hydrogel.
- In one embodiment, the methods according to the present invention do not require the use of expensive oils, chips and/or droplet generation instruments.
- In one embodiment, the methods according to the present invention can be automated.
- In one embodiment, the methods according to the present invention may comprise a step of dissolving the hydrogel matrix. In one embodiment, dissolving of the hydrogel matrix can occur at any time throughout the method. Techniques to dissolve a hydrogel matrix are well-known to the skilled artisan, and comprise, but are not limited to, enzymatic depolymerization using enzymes such as agarase and thermal depolymerization using heat.
- In one embodiment, dissolving of the hydrogel matrix can occur once at least one copy, preferably clonal copies of a unique barcode from at least one barcode unit have been incorporated into the biological unit and/or analytes, such as for example, nucleic acids extracted or derived from a discrete biological unit.
- In one embodiment, depolymerization of the hydrogel matrix can occur once at least one nucleic acid extracted or derived from a discrete biological unit has primed to the at least one oligonucleotide, preferably to the at least one oligonucleotide comprising a nucleic acid sequence primer from a discrete barcode unit.
- The methods described herein can be implemented in a variety of applications, including, but not limited to, single-cell transcriptome profiling, single-cell genotyping, phasing, and single-cell epigenome profiling.
- It will become clear that the embodiments recited in the disclosed applications are not all compulsory features of the present invention, but are only mere illustrations of the implementation of the present invention. The one skilled in the art of single-cell transcriptome profiling, single-cell genotyping, phasing and/or single-cell epigenome profiling will know how to adapt the method using general knowledge of the field. Furthermore, the steps may be combined with and/or modified by any other suitable steps, aspects, and/or features of the present disclosure, including those described in scientific literature and patent documents listed in the present disclosure or known from the skilled artisan.
- The present invention relates to a method for analyzing gene expression in discrete biological units.
- Single-cell transcriptome profiling relies on the amplification of a single cell's mRNAs content and its sequencing. The generation of a single cell transcriptome generally requires a first step of reverse transcription to convert the mRNAs with poly(A) tails into first-strand cDNAs, which can be further amplified and sequenced.
- In one embodiment, the method for analyzing gene expression in discrete biological units may comprise the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed said biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing nucleic acids from each biological unit in the hydrogel matrix,
- e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,
- f) synthetizing a cDNA library from the nucleic acids from each biological unit,
- g) amplifying said cDNA library from each biological unit, wherein amplification of said cDNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, and
- h) sequencing the amplification products.
- In one embodiment, the method for analyzing gene expression in discrete biological units according to the present invention comprises additional steps which are well-known to the skilled artisan. Such steps are described in Macosko et al., 2015. Cell. 161:1202-1214; Fan et al., 2015. Science. 347(6222):1258367; Klein et al., 2015. Cell. 161(5):1187-201; Gierahn et al., 2017. Nαt Methods. 14(4):395-398; and US patent applications
US2016-0289669 ,US2016-0265069 ,US2016-0060621 andUS2015-0376609 . - In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a poly-dT nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a poly-dU nucleic acid sequence primer, a unique barcode and/or a PCR handle.
- In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a (dT)nVN nucleic acid sequence primer, a unique barcode and/or a PCR handle, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e. A, C or G) and N represents any nucleotide (i.e. A, T/U, C or G).
- In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a (dU)nVN nucleic acid sequence primer, a unique barcode and/or a PCR handle, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (i.e. A, C or G) and N represents any nucleotide (i.e. A, T/U, C or G).
- In one embodiment, releasing nucleic acids from each biological unit is performed by cell lysis, preferably by cell lysis using a non-ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of washing out the non-ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of inactivating proteinase K. In one embodiment, inactivation of proteinase K is performed by heat and/or chemical inhibition.
- In one embodiment, synthetizing a cDNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, synthetizing a cDNA library from the nucleic acids from each biological unit is performed by reverse transcription, i.e., using a reverse transcriptase.
- In one embodiment, the reverse transcriptase is a M-MLV reverse transcriptase.
- In one embodiment, a complementary strand of the cDNAs of the cDNA library is synthetized, preferably using second strand reaction components. In one embodiment, the complementary strand of the cDNAs of the cDNA library is synthetized using RNAse H, DNA polymerase I and/or DNA ligase.
- In one embodiment, the cDNA library is fragmented, to obtain cDNA fragments. Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- In one embodiment, cDNA fragments are polished. In one embodiment, cDNA fragments are A-tailed.
- In one embodiment, adaptors are added to the cDNA library. Adaptors may be added to the cDNA library using various methods, including but not limited to, Tn5 transposition and ligation.
- In one embodiment, amplification of the cDNA library is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- The present invention also relates to a method for analyzing the genotype in discrete biological units.
- Single-cell genotyping relies on the whole genome amplification (WGA) of a single cell's DNA to generate enough DNA for sequencing. Several methods for WGA are available and well-known to the skilled artisan. Some methods however lead to amplification bias, and subsequent inadequate genome coverage. PCR-based exponential WGA with degenerate primers introduces sequence-dependent bias. Multiple displacement amplification (MDA), using the strand-displacing Φ29 DNA polymerase, represents an improvement, but may also introduce bias due to nonlinear amplification. Multiple annealing and loop-based amplification cycles (MALBAC) is another method which introduces quasilinear preamplification to reduce the bias associated with nonlinear amplification. It relies on the BstI DNA polymerase for the quasilinear preamplification phase, along with high-fidelity PCR enzymes for subsequent exponential amplification (Zong et al., 2012. Science. 338(6114):1622-6; Lu et al., 2012. Science. 338(6114):1627-30).
- In one embodiment, the method for analyzing the genotype in discrete biological units may comprise the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed said biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing genomic DNA from each biological unit in the hydrogel matrix,
- e) barcoding said genomic DNA from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit,
- g) amplifying said genomic DNA or DNA library from each biological unit, wherein amplification of said genomic DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products of each biological unit, and
- h) sequencing the amplification products.
- In one embodiment, the method for analyzing the genotype in discrete biological units according to the present invention comprises additional steps which are well-known to the skilled artisan. Such steps are described in Hutchison et al., 2005. Proc Natl Acad Sci USA. 102(48):17332-6; Leung et al., 2016. Proc Natl Acad Sci USA. 113(30):8484-9; Wang et al., 2012. Cell. 150(2):402-12; Marcy et al., 2007. PLoS Genet. 3(9):1702-8; Gole et al., 2013. Nat Biotechnol. 31(12):1126-32; Zhang et al., 2006. Nat Biotechnol. 24(6):680-6; and International applications
WO2016/061517 andWO2005/003304 . - In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle. In one embodiment, each barcode unit comprises at least one oligonucleotide comprising an oligo-dN primer (such as an hexanucleotide d(N6) or an octanucleotide d(N8) primer, wherein N represents any nucleotide (i.e., A, T/U, C or G)), a unique barcode and/or a PCR handle.
- In one embodiment, releasing genomic DNA from each biological unit is performed by cell and/or nucleus lysis, preferably by cell and/or nucleus lysis using an ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of washing out the ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of inactivating proteinase K. In one embodiment, inactivation of proteinase K is performed by heat and/or chemical inhibition.
- In one embodiment, the method further comprises a step of denaturation of the genomic DNA. Methods to denature genomic DNA are well-known to the skilled artisan and include, but are not limited to, alkaline treatment and/or heat.
- In one embodiment, synthetizing a cDNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, synthetizing a cDNA library from the nucleic acids from each biological unit is performed by primer-directed extension.
- In one embodiment, amplification of genomic DNA is performed by whole genome amplification (WGA). In one embodiment, amplification of genomic DNA is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, amplified genomic DNA is fragmented, to obtain DNA fragments. Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- In one embodiment, cDNA fragments are polished. In one embodiment, cDNA fragments are A-tailed.
- In one embodiment, adaptors are added to the DNA fragments. Adaptors may be added to the DNA fragments using various methods, including but not limited to, Tn5 transposition and/or ligation.
- In one embodiment, the method for analyzing the genotype in discrete biological units may implement direct library preparation (DLP). In one embodiment, amplified genomic DNA is tagmented. In one embodiment, unamplified genomic DNA is tagmented. Direct library preparation and tagmentation are well-known to the skilled artisan. Reference can be made, e.g., to Vitak et al., 2017. Nat Methods. 14(3):302-308; Adey et al., 2010. Genome Biol. 11(12):R119; Gertz et al., 2012. Genome Res. 22(1):134-41; and Zahn et al., 2017. Nαt Methods. 14(2):167-173. Thus, in one embodiment, each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle. In one embodiment, the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor. In one embodiment, the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2).
- In one embodiment, the method comprises a step of ligating the tagmented genomic DNA from each biological unit to the at least one oligonucleotide of each barcode unit.
- In one embodiment, the method comprises a step of amplification of the DNA fragments. Techniques to amplify DNA fragments are well-known to the skilled artisan.
- In one embodiment, amplification of the DNA fragments is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplification of the DNA fragments is performed with at least one nucleic acid sequence primer which is not the at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- The present invention also relates to a method for analyzing the haplotype of discrete biological units, i.e., for phasing.
- Phasing relies on the whole genome amplification (WGA) of a high molecular weight, i.e., greater than 25 or 50 kilobases, DNA to generate enough DNA for sequencing. Several methods for WGA are available and well-known to the skilled artisan. Some methods however lead to amplification bias, and subsequent inadequate genome coverage. PCR-based exponential WGA with degenerate primers introduces sequence-dependent bias. Multiple displacement amplification (MDA), using the strand-displacing Φ29 DNA polymerase, represents an improvement, but may also introduce bias due to nonlinear amplification. Multiple annealing and loop-based amplification cycles (MALBAC) is another method which introduces quasilinear preamplification to reduce the bias associate with nonlinear amplification. It relies on the BstI DNA polymerase for the quasilinear preamplification phase, along with high-fidelity PCR enzymes for subsequent exponential amplification (Zong et al., 2012. Science. 338(6114):1622-6; Lu et al., 2012. Science. 338(6114):1627-30). Alternatively, the Tn5 transposase and subsequent amplification can be used for library prep in a method termed "Contiguity-Preserving Transposition" (CPT-seq) (Amini et al., 2014. Nαt Genet. 46(12):1343-9). In this method, the first step after the genomic DNA has been optionally purified is to tagment the DNA through Tn5 transposition. This fragments the DNA and adds universal adaptors directly to the template. After gap filling, PCR then occurs using primers complementary to the inserted Tn5 adaptors followed by sequencing.
- In one embodiment, the method for analyzing the haplotype of discrete biological units may comprise the steps of:
- a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed said biological unit/barcode unit complexes in a hydrogel matrix,
- d) optionally, releasing nucleic acids from each biological unit in the hydrogel matrix,
- e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit,
- g) amplifying said nucleic acid or DNA library from each biological unit, wherein amplification of said nucleic acids or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, and
- h) sequencing the amplification products.
- In one embodiment, the method for analyzing the haplotype in discrete biological units according to the present invention comprises additional steps which are well-known to the skilled artisan. Such steps are described in International applications
WO2015/126766 ,WO2016/130704 ,WO2016/61517 WO2015/95226 WO2016/003814 ,WO2005/003304 ,WO2015/200869 ,WO2014/124338 ,WO2014/093676 ;US patent application US2015-066385 ; Kuleshov et al., 2014. Nαt Biotechnol. 32(3):261-6; Amini et al., 2014. Nat Genet. 46(12):1343-9; Kaper et al., 2013. Proc Natl Acad Sci USA. 110(14):5552-7; Peters et al., 2012. Nature. 487(7406):190-5 and Zheng et al., 2016. Nat Biotechnol. 34(3):303-11. - In one embodiment, the at least one means for binding a biological unit is an anti-Tn5 antibody. In one embodiment, the at least one means for binding a biological unit is streptavidin and the biological unit is contacted with a biotinylated anti-Tn5 antibody.
- In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle. In one embodiment, the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor. In one embodiment, the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2). In another embodiment, each barcode unit comprises at least one oligonucleotide comprising an oligo-dN primer (such as an hexanucleotide d(N6) or an octanucleotide d(N8) primer, wherein N represents any nucleotide (i.e., A, T/U, C or G)), a unique barcode and/or a PCR handle. In one embodiment, releasing nucleic acids from each biological unit is performed by cell and/or nucleus lysis, preferably by cell and/or nucleus lysis using an ionic detergent and/or proteinase K.
- In one embodiment, synthetizing a DNA library from the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, the method further comprises a step of washing out the ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of inactivating proteinase K. In one embodiment, inactivation of proteinase K is performed by heat and/or chemical inhibition.
- In one embodiment, the method further comprises a step of denaturation of the nucleic acids from each biological unit. Methods to denature nucleic acids are well-known to the skilled artisan and include, but are not limited to, alkaline treatment and/or heat.
- In one embodiment, amplification of the nucleic acids from each biological unit is performed by whole genome amplification (WGA). In one embodiment, amplification of the nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplified nucleic acids from each biological unit are fragmented, to obtain nucleic acid fragments. Methods for fragmenting DNA are well-known in the art, and include, but are not limited to, Covaris sonication and DNA enzymatic cutting.
- In one embodiment, nucleic acid fragments are polished. In one embodiment, nucleic acid fragments are A-tailed.
- In one embodiment, adaptors are added to the nucleic acid fragments, preferably Tn5 adaptors. Adaptors may be added to the nucleic acid fragments using various methods, including but not limited to, Tn5 transposition and ligation.
- In one embodiment, the method for analyzing the haplotype in discrete biological units may implement contiguity-preserving transposition (CTP-seq). Such method is described in international application
WO2016/061517 . - In one embodiment, the method comprises a step of tagmenting nucleic acids from each biological unit, preferably with Tn5 transposase. In one embodiment, nucleic acids from each biological unit are high molecular weight DNA (HMW-DNA). In one embodiment, tagmenting HMW-DNA from each biological unit preserves the contiguity of the HMW-DNA from each biological.
- In one embodiment, the method comprises a step of disrupting contiguity of the nucleic acids from each biological unit, preferably of the HMW-DNA from each biological unit. Techniques to disrupt contiguity are well-known to the skilled artisan and include, but are not limited to, release of Tn5 complexes from the nucleic acids from each biological unit, preferably by using an ionic detergent and/or proteinase K.
- In one embodiment, the method comprises a step of gap filling of the adaptor, preferably of the Tn5 adaptor. In one embodiment, the method comprises a step of amplification of the tagmented nucleic acids from each biological unit. In one embodiment, amplification of the tagmented nucleic acids from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, the method comprises a step of ligating the tagmented nucleic acids from each biological unit to the at least one oligonucleotide of each barcode unit.
- In one embodiment, the method comprises a step of amplification of the tagmented nucleic acids. Techniques to amplify of nucleic acids are well-known to the skilled artisan. In one embodiment, amplification of the tagmented nucleic acids is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
- The present invention also relates to a method for analyzing the epigenome in discrete biological units.
- Single cell nucleosome positioning based on Tn5 transposition has been developed, termed "Assay for Transposase-Accessible Chromatin with high throughput sequencing" (ATAC-seq) (Buenrostro et al., 2015. Nature. 523(7561):486-90). In this method, the first step enable molecular access to nucleosome-free DNA by using low percentage non-ionic detergents on intact cells or isolated nuclei. The accessible DNA is then tagmented through Tn5 transposition. This fragments the DNA and adds universal adaptors directly to the template. PCR then occurs using primers complementary to those adaptors followed by sequencing.
- Thus, in one embodiment, the method for analyzing the epigenome in discrete biological units may comprise the steps of:
- a) contacting a plurality of cellular biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,
- b) contacting said biological unit/barcode unit complexes with a hydrogel solution,
- c) polymerizing the hydrogel solution to embed said biological unit/barcode unit complexes in a hydrogel matrix,
- d) releasing non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,
- e) barcoding said non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,
- f) optionally, synthetizing a DNA library from the non-nucleosome bound DNA from each biological unit,
- g) amplifying said non-nucleosome-bound-DNA or DNA library from each biological unit, wherein amplification of said non-nucleosome-bound-DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit,
- h) sequencing the amplification products.
- In one embodiment, amplification of non-nucleosome-bound-DNA or DNA library from each biological unit starts from non-nucleosome start sites. Non-nucleosome start sites are sites where transposition occurs, i.e., where the DNA is accessible. Optionally, non-nucleosome start sites are sites where DNA is enzymatically fragmented and where DNA is ligated.
- In one embodiment, the method for analyzing the epigenome in discrete biological units according to the present invention comprises additional steps which are well-known to the skilled artisan. Such steps are described in International application
WO2014/189957 ; Buenrostro et al., 2015. Nature. 523(7561):486-90; Buenrostro et al., 2013. Nαt Methods. 10(12):1213-8; and Christiansen et al., 2017. Methods Mol Biol. 1551:207-221. - In one embodiment, each barcode unit comprises at least one oligonucleotide comprising a nucleic acid sequence primer, a unique barcode and/or a PCR handle. In one embodiment, the nucleic acid sequence primer has a sequence which is complementary to at least one adaptor sequence, preferably at least one Illumina adaptor sequence. In one embodiment, the nucleic acid sequence primer has a sequence which is complementary to at least one Tn5 adaptor. In one embodiment, the nucleic acid sequence primer comprises or consist of sequence 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 1) or 5'-GTCTCGTGGGCTCG-3' (SEQ ID NO: 2).
- In one embodiment, releasing non-nucleosome bound DNA from each biological unit is performed by cell lysis, preferably by cell lysis using a non-ionic detergent and/or proteinase K.
- In one embodiment, synthetizing a DNA library from the non-nucleosome bound DNA from each biological unit is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, the method further comprises a step of washing out the non-ionic detergent and/or proteinase K.
- In one embodiment, the method further comprises a step of inactivating proteinase K. In one embodiment, inactivation of proteinase K is performed by heat and/or chemical inhibition.
- In one embodiment, non-nucleosome bound DNA is tagmented. Techniques for tagmentation are well-known to the skilled artisan. In one embodiment, tagmentation of non-nucleosome bound DNA is performed by Tn5 transposition, preferably using Illumina adaptor sequences.
- In one embodiment, the method comprises a step of ligating the tagmented non-nucleosome bound DNA from each biological unit to the at least one oligonucleotide of each barcode unit.
- In one embodiment, the method comprises a step of amplification of the tagmented non-nucleosome bound DNA from each biological unit. Techniques to amplify DNA are well-known to the skilled artisan.
- In one embodiment, amplification of the tagmented non-nucleosome bound DNA is performed with at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit. In one embodiment, amplification of the tagmented non-nucleosome bound DNA is performed with at least one nucleic acid sequence primer which is not the at least one nucleic acid sequence primer of the at least one oligonucleotide of the barcode unit.
- In one embodiment, amplification of the tagmented non-nucleosome bound DNA incorporates the adaptor sequence from the Tn5 transposases into the amplification products from each biological unit.
- In one embodiment, amplification steps can be enhanced using free nucleic acid sequence primers, i.e., nucleic acid sequence primer which are not bound to a barcode unit.
-
-
Figure 1 is a diagram illustrating the trapping and barcoding of biological units in hydrogel. The following symbols are used: (A) Barcode unit; (B) Biological unit; (B*) Barcoded biological unit; (C) Means for binding biological units; (Hs) Hydrogel (sol state); (He) Hydrogel matrix (hydrogel in gel state); (HG /HS ) Hydrogel in solid or gel state; (1) Binding of biological units and barcode units; (2) Contacting with hydrogel solution; (3) Polymerization of hydrogel; (4) Barcoding of biological units; (5) Primer-directed extension, Ligation, Amplification, Fragmentation, Adaptering; (6) Next generation sequencing. -
Figure 2 is a diagram illustrating multiple biological units binding to a single barcode unit. The following symbols are used: (A1, A2) Barcode units; (B1, B2) Biological units; (C) Means for binding biological units; ( Y ) Biased data; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 3 is a diagram illustrating multiple barcode units binding to a single biological unit. The following symbols are used: (A1, A2) Barcode units; (B1, B2) Biological units; (C) Means for binding biological units; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 4 is a diagram illustrating the binding of biological units to a solid support before binding to barcode units, trapping, and barcoding. Barcode units are significantly larger than biological units, preventing therefore the binding of multiple barcode units to a single biological unit. The following symbols are used: (A1, A2) Barcode units; (B1, B2) Biological units; (C) Means for binding biological units; (S) Solid support; (11) Binding of biological units to solid support; (12) Addition of barcode units in solution; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 5 is a diagram illustrating the binding of barcode units to a solid support before binding to biological units, trapping, and barcoding. Biological units are significantly larger than barcode units, preventing therefore the binding of multiple biologic units to a single barcode unit. The following symbols are used: (A1, A2) Barcode units; (B1, B2) Biological units; (C) Means for binding biological units; (D) Means for binding barcode units; (S) Solid support; (21) Binding of barcode units to solid support; (22) Addition of biological units in solution; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 6 is a diagram illustrating the binding of biological units to a solid support before binding to barcode units, trapping, and barcoding. Barcode units and biological units are roughly the same size. Barcode units are at limiting dilution to preventing the binding of multiple barcode units to a single biological unit. The following symbols are used: (A) Barcode unit; (B1, B2) Biological units; (C) Means for binding biological units; (S) Solid support; (11) Binding of biological units to solid support; (12*) Addition of barcode units in solution at a limiting concentration; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 7 is a diagram illustrating the binding of barcode units to a solid support before binding to biological units, trapping, and barcoding. Biological units and barcode units are roughly the same size. Biological units are at limiting dilution to preventing the binding of multiple biological units to a single barcode unit. The following symbols are used: (A1, A2) Barcode units; (B) Biological unit; (C) Means for binding biological units; (D) Means for binding barcode units; (S) Solid support; (21) Binding of barcode units to solid support; (22*) Addition of biological units in solution at a limiting concentration; (1) Binding of biological units and barcode units; (2-6) Steps 2 to 6 ofFigure 1 . -
Figure 8 is a diagram illustrating a possible single cell RNAseq transcriptome workflow, using barcode units comprising an oligonucleotide, itself comprising a poly-dT nucleic acid sequence primer, a unique barcode and a PCR handle. Multiple barcode oligonucleotides are present from the first step, but only one is shown here, as (a), afterstep 84 for simplicity. Steps 1-3 (1-3) may be performed as inFigure 1 or may involve a solid support and include therefore the additional steps ofFigures 4 to 7 . The following symbols are used: (A) Barcode unit; (B) Biological unit; (HG ) Hydrogel matrix (hydrogel in gel state); (HG /HS ) Hydrogel in solid or gel state; (R) Poly(A) mRNA; (a) barcode; (PCR) PCR handle; (Tn ) Poly(T) primer; (DNA1) First strand cDNA; (DNA2) 2nd strand cDNA; (83*) Cell lysis by application of a non-ionic detergent; (84) Barcoding, i.e., priming of poly(A) mRNAs with oligo d(T) primer of barcode oligonucleotides; (85) 2nd strand cDNA synthesis (optionally through template switching and amplification); (86) Fragmentation, Adaptering, Amplification, Next-Generation sequencing. -
Figure 9 is a diagram illustrating a possible phasing workflow, using barcode units comprising an oligonucleotide, itself comprising a complementary Tn5 adaptor nucleic acid sequence primer, a unique barcode and a PCR handle. Multiple barcode oligonucleotides are present from the first step, but only one is shown here afterstep 94 for simplicity. Binding to a solid support of the barcode unit as infigures 5 and7 or of the transposases as inFigures 4 to 6 is possible. The following symbols are used: (A) Barcode unit; (CPT) Contiguity-preserved transposition DNA; (Tn5) Tn5 transposase; (Tn5s) Tn5 adaptor sequence; (a) barcode; (PCR) PCR handle; (Tn5P ) Tn5 adaptor primer; (Hs) Hydrogel (sol state); (He) Hydrogel matrix (hydrogel in gel state); (HG /HS ) Hydrogel in solid or gel state; (91) Binding transposase to barcode unit; (2) Contacting with hydrogel solution; (3) Polymerization of hydrogel; (94) Release transposase; (95) Ligation, Gap-filling; (96) Amplification, Next-Generation sequencing. - The present invention is illustrated by the following examples. However, it should be understood that the invention is not limited to the specific details of these examples.
- The present invention relates to the trapping of discrete biological units (i.e., cells or groups of cells, viruses, organelles, macromolecular complexes or biological macromolecules).
- The present invention and its applications rests upon the implementation of successive steps described in
Figure 1 . - In a first step, biological unit/barcode unit complexes are formed, each complex comprising a single barcode unit and a single biological unit (
step 1 ofFigure 1 ). Biological unit/barcode unit complexes can be formed upon binding and/or immobilization of the biological unit on the barcode unit. Barcode units must thus carry on their surface a means for binding, either specifically or non-specifically, biological units. These means include proteins or fragments thereof, peptides, antibodies or fragments thereof, nucleic acids, carbohydrates, vitamins or derivatives thereof, coenzymes or derivative thereof, receptor ligands derivative thereof and/or hydrophobic groups. Concurrently, the biological units must carry, either naturally or not, a complementary means, binding to the means of the barcode unit. For example, a means for binding a biological unit can be an antibody, directed to molecules expressed or present (either naturally or artificially) at the surface of the biological unit. Another option can be the use of a biotinylated antibody directed to molecules expressed or present at the surface of the biological unit, and the subsequent binding of the biological unit carrying the biotinylated antibody to barcode units coated with streptavidin. - Once the biological unit/barcode unit complexes are formed, they can be contacted with a hydrogel solution, which upon polymerization, traps the biological unit/barcode unit complexes (steps 2-3 of
Figure 1 ). Biochemistry and molecular biology assays can then be performed directly in the hydrogel matrix, by contacting the hydrogel with any required reagent and/or solution. - For example, a suitable hydrogel solution can be alginate. Its fine grain size allows for the formation of very small pores upon polymerization with calcium, trapping the biological unit/barcode unit complexes without any risk of diffusion, while still allowing for the diffusion of smaller components like reagent and/or solution. Typically, when the biological unit is a cell, a group of cell, a nucleus or an organelle, a first step will comprise the lysis of the biological unit, to release its nucleic acid content. Any detergent level is supported by the hydrogel platform, allowing to lyse even difficult-to-lyse biological units.
- The released nucleic acids can then be barcoded (
step 4 ofFigure 1 ), through priming to the oligonucleotide coated on the surface of the barcode unit. Typically, each barcode unit comprises clonal copies of an oligonucleotide, which is composed of at least one priming site (nucleic acid sequence primer) and a barcode sequence. The barcode sequence should always be identical in every oligonucleotide of a given barcode unit, so as to allow identification of the source or origin of the nucleic acids extracted or derived from one discrete biological unit. - Once barcoding is achieved (i.e., priming of the biological unit's nucleic acids to the barcode unit's nucleic acid sequence primer), classical biochemistry and molecular biology assays can be carried out on the barcoded nucleic acids, either while still entrapped in the hydrogel matrix, or in solution, after hydrogel matrix has been dissolved. These include without limitation and not necessarily in this order, primer-directed extension, ligation, amplification, fragmentation, addition of adaptor sequences, next generation sequencing and the like (steps 5-6 of
Figure 1 ). For example, when using alginate as a hydrogel, calcium can be washed out from the hydrogel to allow depolymerization. Stabilization of the primed, i.e., barcoded nucleic acids, prior to any biochemistry and molecular biology assay, and in particular, prior to primer-directed extension, can be achieved using other cations, such as sodium. - A crucial step when implementing the method of the present invention is the binding of a single biological unit to a single barcode unit, as to form a 1:1 complex. As shown in
Figure 2 , the binding of multiple biological units to a single barcode unit skews the subsequent data retrieved, and in particular, single cell next generation sequencing data. Upon sequence analysis, sequences with "barcode 1" would be biased or corrupted since they are gathered from two distinct biological units. - Likewise, the binding of multiple barcode units to a single biological unit skews the single cell next generation sequencing data (
Figure 3 ). Sequence data gathered from "biological unit 1" (B1) would be represented twice by "barcode 1" and "barcode 2" (A1 and A2). - Several ways can help avoiding the formation of non-stoichiometric biological unit/barcode unit complexes.
-
Figure 4 shows the immobilization of the biological units of interest on a support, coated with means for binding said biological units (step 11). Once immobilization on the support, biological units can be contacted with barcode units (step 12) - preferentially with barcode units which are larger in size with respect to the biological units, to create hindrance and prevent the binding of multiple barcode unit on a single biological unit (step 1). Therefore, since only one barcode unit is bound per biological unit, it is possible to parse subsequent next generation sequencing data into single biological units. - Such configuration can be easily implemented, using a support such as a microcentrifuge tube coated with a means for binding biological units, such as biotin. Biological units such as cells are contacted with streptavidin-coupled antibodies, then deposited in the tube to allow for binding. Excess cells are removed. Biotin-coated barcode units, such as beads, are then deposited in the tube to allow for binding to the cells. Excess beads are removed. A hydrogel solution is then poured into the tube, such as sodium alginate, together with calcium ions, to allow alginate to polymerize. Trapped cells can then be processed, such as for example by addition of detergent on top of the tube. By capillarity, the detergent reaches the trapped cells and lyse their membrane, releasing their nucleic acid content. Alginate pore size is small enough to avoid diffusion of nucleic acids, while allowing diffusion of smaller reactants and substrates. Barcoding occurs as nucleic acids from a discrete cell are released and attach to the nucleic acid sequence barcode of their adjacent barcode bead. Once the nucleic acids are properly barcoded, the sample can be wash out to remove calcium ions. Alginate hydrogel dissolves, and further steps can be processed directly in the tube, in solution.
- Alternatively, barcode units can be bound on a support, coated with means for binding said barcode units. Once bound to the support, barcode units can be contacted with biological units - preferentially with biological units which are larger in size with respect to the barcode units, to create hindrance and prevent the binding of multiple biological units on a single barcode unit (
Figure 5 ). - Such configuration can also be implemented using a support such as a microcentrifuge tube coated with a thin layer of hydrogel which, upon polymerization, immobilizes barcode units throughout the support. Biological units such as cells are then deposited in the tube to allow for binding to the barcode units (providing that the layer of hydrogel immobilizing the barcode units is thinner than the smallest dimension of the barcode unit, i.e., that at least a part of the barcode unit remains accessible for contacting biological units). Excess cells are removed. A hydrogel solution is then poured into the tube and left polymerizing. Trapped cells can then be processed as described hereinabove. Once the nucleic acids are properly barcoded, both hydrogels (i.e., the thin layer coating the tube and the hydrogel matrix trapping the biological units) can be dissolved, and further steps can be processed directly in the tube, in solution.
- Another strategy to avoid the formation of non-stoichiometric biological unit/barcode unit complexes is the use of a support where biological units of interest (
Figure 6 ) or barcode units (Figure 7 ) are bound and/or immobilized as described previously, together with limiting concentrations of barcode units or biological units, respectively. Preferably, the concentration of free units (barcode units or biological units, respectively) is lower than the concentration of support-bound units (biological units or barcode units, respectively). This ensures the binding of at most one barcode unit per biological unit and conversely, making it possible to parse subsequent next generation sequencing data into single biological units. Some biological units (step 1 ofFigure 6 ) or barcode units (step 1 ofFigure 7 ) are not coupled with a barcode unit or a biological unit, respectively, and therefore do not produce any data. - Single-cell transcriptome profiling is one of the numerous biochemistry and molecular biology assays that can be carried out using the method of the present invention (
Figure 8 ). - After forming biological unit/barcode unit complexes in a hydrogel solution as described in Example 1 (steps 1-3 of
Figure 1 ; optionally after the additional steps (11 and 12 or 12*, or 21 and 22 or 22*) of any ofFigures 4-7 , the hydrogel is allowed to polymerize, trapping thus biological unit/barcode unit complexes (" 1-3" inFigure 8 ). - Most commonly, the biological units will be a cell, such as a mammalian cell for example, or any other cell suitable for single-cell transcriptome profiling. Single-cell transcriptome profiling relies on the amplification of a single cell's mRNAs content and its sequencing. A first step is therefore to release the cells' mRNAs content, by lysing the cells directly in the hydrogel. To do so, non-ionic detergents or any other suitable reagent for cell lysis can be applied directly on the hydrogel matrix. By diffusion, the reagent can reach up to the biological units, and lyse them (step 83* of
Figure 8 ). - The released mRNAs bind in their local environment to the oligonucleotides carried by the barcode units. These oligonucleotides are present in multiple clonal copies on each barcode unit, and are unique as to their sequence from barcode unit to barcode unit. They comprise a PCR handle, a unique barcode sequence and a nucleic acid sequence primer.
- Mammalian mRNAs possess a natural 3' poly(A) sequence, which can therefore prime to a nucleic acid sequence primer comprising a poly(T) sequence (step 84 of
Figure 8 ). Upon priming (i.e., barcoding), the following molecular biology steps can take place either within the hydrogel matrix or in solution. Typically, first-strand cDNA synthesis will occur in 3' of the barcode unit oligonucleotide, using a reverse transcriptase enzyme. - Second strand cDNA synthesis can then occur, optionally through template switching and amplification (
step 85 ofFigure 8 ). Next steps comprise for example fragmentation of the cDNA library, adaptering, and amplification. - Barcoded, amplified and adaptered products can finally be sequenced by next generation sequencing (
step 86 ofFigure 8 ). - Phasing is another molecular biology assay that can be carried out using the method of the present invention (
Figure 9 ). - In a first step, transposomes are assembled in solution by mixing a Tn5 transposase with high molecular weight DNA (i.e., the biological unit). This step, sometimes referred to as tagmentation, creates contiguity preserved transposition DNA (CPT-DNA) fragments, and is followed by a second step wherein the transposomes are contacted with barcode units, comprising a means for binding the biological unit (step 91 of
Figure 9 ). Advantageously, this means binds Tn5 transposases. - The CPT-DNA/barcode unit complexes are then contacted with a hydrogel solution, which is left to polymerize (steps 2-3 of
Figure 9 ). Once trapped in the hydrogel matrix, the Tn5 transposases are released, using ionic detergents and/or proteinase K, disrupting thus contiguity and yielding DNA fragments comprising a Tn5 adaptor sequence (step 94 ofFigure 9 ). - The released DNA fragments, comprising a Tn5 adaptor sequence, can prime in their local environment to a nucleic acid sequence primer carried by the barcode units, and comprising a complementary Tn5 adaptor sequence (such as, e.g., SEQ ID NO: 1 or SEQ ID NO: 2). These oligonucleotides are present in multiple clonal copies on each barcode unit, and are unique as to their sequence from barcode unit to barcode unit. They comprise a PCR handle, a unique barcode sequence and a nucleic acid sequence primer, complementary to the Tn5 adaptor sequence (Tn5 adaptor primer, Tn5P). Upon priming (i.e., barcoding), the following molecular biology steps can take place either within the hydrogel matrix or in solution, upon dissolving of the hydrogel. Ligation, gap-filling and amplification (
step 95 ofFigure 9 ), can occur either in the hydrogel matrix or in solution. - Barcoded, amplified and adaptered products can finally be sequenced by next generation sequencing (
step 96 ofFigure 9 ). - Other variations of molecular biology can be found in international patent application
WO2016/061517 (e.g., in Figures 15-21).
Claims (13)
- A method for trapping discrete biological units in a hydrogel, said method comprising the steps of:a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,b) contacting said biological unit/barcode unit complexes with a hydrogel solution, andc) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,d) barcoding the biological unit's nucleic acid within each of said biological unit/barcode unit complexes in the hydrogel matrix.
- A method for analyzing gene expression in discrete biological units, said method comprising the steps of:a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,b) contacting said biological unit/barcode unit complexes with a hydrogel solution,c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,d) releasing nucleic acids from each biological unit in the hydrogel matrix,e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,f) synthetizing a cDNA library from the nucleic acids from each biological unit,g) amplifying said cDNA library from each biological unit, wherein amplification of said cDNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, andh) optionally, sequencing the amplification products.
- A method for analyzing the genotype in discrete biological units, said method comprising the steps of:a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,b) contacting said biological unit/barcode unit complexes with a hydrogel solution,c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,d) releasing genomic DNA from each biological unit in the hydrogel matrix,e) barcoding said genomic DNA from each biological unit in the hydrogel matrix,f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit,g) amplifying said genomic DNA or DNA library from each biological unit, wherein amplification of said genomic DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products of each biological unit, andh) optionally, sequencing the amplification products.
- A method for analyzing the haplotype of discrete biological units, said method comprising the steps of:a) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,b) contacting said biological unit/barcode unit complexes with a hydrogel solution,c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,d) optionally, releasing nucleic acids from each biological unit in the hydrogel matrix,e) barcoding said nucleic acids from each biological unit in the hydrogel matrix,f) optionally, synthetizing a DNA library from the nucleic acids from each biological unit,g) amplifying said nucleic acid or DNA library from each biological unit, wherein amplification of said nucleic acids or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, andh) optionally, sequencing the amplification products.
- A method for analyzing the epigenome in discrete biological units, said method comprising the steps of:a) contacting a plurality of cellular biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units,b) contacting said biological unit/barcode unit complexes with a hydrogel solution,c) polymerizing the hydrogel solution to embed discrete biological unit/barcode unit complexes in a hydrogel matrix,d) releasing non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,e) barcoding said non-nucleosome-bound-DNA from each biological unit in the hydrogel matrix,f) optionally, synthetizing a DNA library from the non-nucleosome bound DNA from each biological unit,g) amplifying said non-nucleosome-bound-DNA or DNA library from each biological unit, wherein amplification of said non-nucleosome-bound-DNA or DNA library from each biological unit incorporates clonal copies of said unique barcode into the amplification products from each biological unit, andh) optionally, sequencing the amplification products.
- The method according to claim 1, 2, 3, 4 or 5, wherein:- said biological units are immobilized on a support; or- said barcode units are immobilized on a support.
- The method according to claim 6, wherein said biological units or said barcode units are immobilized on a support in a hydrogel layer.
- The method according to any one of claims 1 to 7, wherein said unique barcode is present in multiple clonal copies on each barcode unit.
- The method according to any one of claims 1 to 8, wherein said unique barcode comprises a nucleic acid sequence barcode.
- The method according to any one of claims 1 to 9, wherein said unique barcode further comprises a nucleic acid sequence primer.
- The method according to any one of claims 1 to 10, wherein said each barcode unit consists of a bead.
- The method according to any one of claims 1 to 11, wherein said discrete biological units comprise cells, groups of cells, viruses, nuclei, mitochondria, chloroplasts, biological macromolecules, exosomes, chromosomes, contiguity preserved transposition DNA fragments and/or nucleic acid fragments.
- The method according to any one of claims 1 to 12, wherein said at least one means involved with binding said biological units comprise a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid, a carbohydrate, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, or a hydrophobic group.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP24150478.6A EP4345159A2 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762502180P | 2017-05-05 | 2017-05-05 | |
PCT/IB2018/000612 WO2018203141A1 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP24150478.6A Division EP4345159A2 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
Publications (3)
Publication Number | Publication Date |
---|---|
EP3619325A1 EP3619325A1 (en) | 2020-03-11 |
EP3619325B1 true EP3619325B1 (en) | 2024-01-24 |
EP3619325C0 EP3619325C0 (en) | 2024-01-24 |
Family
ID=62705617
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP24150478.6A Pending EP4345159A2 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
EP18733327.3A Active EP3619325B1 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP24150478.6A Pending EP4345159A2 (en) | 2017-05-05 | 2018-05-04 | Methods for trapping and barcoding discrete biological units in hydrogel |
Country Status (10)
Country | Link |
---|---|
US (1) | US20180320173A1 (en) |
EP (2) | EP4345159A2 (en) |
JP (2) | JP7197567B2 (en) |
KR (1) | KR20200004335A (en) |
CN (1) | CN111148846A (en) |
AU (1) | AU2018262331A1 (en) |
CA (1) | CA3062248A1 (en) |
GB (1) | GB2577214B (en) |
SG (1) | SG11201910195WA (en) |
WO (1) | WO2018203141A1 (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201622222D0 (en) | 2016-12-23 | 2017-02-08 | Cs Genetics Ltd | Reagents and methods for molecular barcoding of nucleic acids of single cells |
WO2021011895A1 (en) * | 2019-07-18 | 2021-01-21 | Celldom, Inc. | Methods and devices for single cell barcoding |
WO2021116371A1 (en) * | 2019-12-12 | 2021-06-17 | Keygene N.V. | Semi-solid state nucleic acid manipulation |
WO2023139272A1 (en) | 2022-01-24 | 2023-07-27 | Scipio Bioscience | Gelation device with piston |
EP4272764A1 (en) | 2022-05-03 | 2023-11-08 | Scipio Bioscience | Method of complexing biological units with particles |
WO2024013218A1 (en) | 2022-07-12 | 2024-01-18 | Paris Sciences Et Lettres | Method for spatial tracing and sequencing of cells or organelles |
CN115463622A (en) * | 2022-08-03 | 2022-12-13 | 广东纤友朵美生物科技有限公司 | Gel based on oxidized pectin and preparation method thereof |
Family Cites Families (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2004253882B2 (en) | 2003-06-20 | 2010-06-10 | Illumina, Inc. | Methods and compositions for whole genome amplification and genotyping |
EP2324045A4 (en) * | 2008-08-05 | 2013-04-03 | Univ Cornell | Photo-crosslinked nucleic acid hydrogels |
GB2497912B (en) * | 2010-10-08 | 2014-06-04 | Harvard College | High-throughput single cell barcoding |
WO2013116698A2 (en) * | 2012-02-02 | 2013-08-08 | Invenra, Inc. | High throughput screen for biologically active polypeptides |
CN113528634A (en) * | 2012-08-14 | 2021-10-22 | 10X基因组学有限公司 | Microcapsule compositions and methods |
EP3567116A1 (en) | 2012-12-14 | 2019-11-13 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
CA2900543C (en) | 2013-02-08 | 2023-01-31 | 10X Genomics, Inc. | Partitioning and processing of analytes and other species |
CN111394426A (en) | 2013-05-23 | 2020-07-10 | 斯坦福大学托管董事会 | Transposition to native chromatin for personal epigenomics |
DK3039158T3 (en) | 2013-08-28 | 2019-03-04 | Becton Dickinson Co | MASSIVE PARALLEL SINGLE CELL CELL ANALYSIS |
US10395758B2 (en) | 2013-08-30 | 2019-08-27 | 10X Genomics, Inc. | Sequencing methods |
US9709479B2 (en) * | 2013-10-25 | 2017-07-18 | Massachusetts Institute Of Technology | Method and apparatus for tracking cell identity |
DK3083994T3 (en) | 2013-12-20 | 2021-09-13 | Illumina Inc | Preservation of genomic connectivity information in fragmented genomic DNA samples |
AU2014373757B2 (en) * | 2013-12-30 | 2019-12-12 | Atreca, Inc. | Analysis of nucleic acids associated with single cells using nucleic acid barcodes |
CA2940048C (en) | 2014-02-18 | 2023-03-14 | Illumina, Inc. | Methods and compositions for dna profiling |
CN114214314A (en) * | 2014-06-24 | 2022-03-22 | 生物辐射实验室股份有限公司 | Digital PCR barcoding |
MX2016016904A (en) | 2014-06-26 | 2017-03-27 | 10X Genomics Inc | Analysis of nucleic acid sequences. |
EP3161160B1 (en) * | 2014-06-26 | 2021-10-13 | 10X Genomics, Inc. | Methods of analyzing nucleic acids from individual cells or cell populations |
WO2016003814A1 (en) | 2014-06-30 | 2016-01-07 | Illumina, Inc. | Methods and compositions using one-sided transposition |
SG11201703139VA (en) | 2014-10-17 | 2017-07-28 | Illumina Cambridge Ltd | Contiguity preserving transposition |
EP3248018B1 (en) | 2015-01-22 | 2020-01-08 | Becton, Dickinson and Company | Devices and systems for molecular barcoding of nucleic acid targets in single cells |
ES2786652T3 (en) * | 2015-02-10 | 2020-10-13 | Illumina Inc | Methods and compositions for analyzing cellular components |
WO2017075265A1 (en) * | 2015-10-28 | 2017-05-04 | The Broad Institute, Inc. | Multiplex analysis of single cell constituents |
-
2018
- 2018-05-04 US US15/971,417 patent/US20180320173A1/en active Pending
- 2018-05-04 WO PCT/IB2018/000612 patent/WO2018203141A1/en active Application Filing
- 2018-05-04 GB GB1917721.1A patent/GB2577214B/en active Active
- 2018-05-04 CN CN201880041302.XA patent/CN111148846A/en active Pending
- 2018-05-04 SG SG11201910195W patent/SG11201910195WA/en unknown
- 2018-05-04 AU AU2018262331A patent/AU2018262331A1/en active Pending
- 2018-05-04 CA CA3062248A patent/CA3062248A1/en active Pending
- 2018-05-04 KR KR1020197034770A patent/KR20200004335A/en not_active Application Discontinuation
- 2018-05-04 JP JP2020512098A patent/JP7197567B2/en active Active
- 2018-05-04 EP EP24150478.6A patent/EP4345159A2/en active Pending
- 2018-05-04 EP EP18733327.3A patent/EP3619325B1/en active Active
-
2022
- 2022-12-15 JP JP2022200037A patent/JP2023030031A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
AU2018262331A1 (en) | 2019-11-21 |
US20180320173A1 (en) | 2018-11-08 |
JP2020518292A (en) | 2020-06-25 |
GB201917721D0 (en) | 2020-01-15 |
CN111148846A (en) | 2020-05-12 |
EP3619325A1 (en) | 2020-03-11 |
KR20200004335A (en) | 2020-01-13 |
SG11201910195WA (en) | 2019-11-28 |
WO2018203141A1 (en) | 2018-11-08 |
JP2023030031A (en) | 2023-03-07 |
AU2018262331A8 (en) | 2019-12-12 |
JP7197567B2 (en) | 2022-12-27 |
EP4345159A2 (en) | 2024-04-03 |
EP3619325C0 (en) | 2024-01-24 |
GB2577214A (en) | 2020-03-18 |
GB2577214B (en) | 2021-10-06 |
CA3062248A1 (en) | 2018-11-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3619325B1 (en) | Methods for trapping and barcoding discrete biological units in hydrogel | |
US20210380974A1 (en) | Combinatorial sets of nucleic acid barcodes for analysis of nucleic acids associated with single cells | |
EP3752634B1 (en) | Dna sequencing using hydrogel beads | |
EP3244992B1 (en) | Processes for barcoding nucleic acids | |
US20180312833A1 (en) | Sample preparation on a solid support | |
US20220411859A1 (en) | Hydrogel beads for nucleotide sequencing | |
JP2020505003A (en) | Kinetic exclusion amplification of nucleic acid libraries | |
CN114854832A (en) | Nucleic acid amplification | |
US20200131502A1 (en) | Modulating polymer beads for dna processing | |
US20210163926A1 (en) | Versatile amplicon single-cell droplet sequencing-based shotgun screening platform to accelerate functional genomics | |
JP2014533096A (en) | Method for spatial placement of sample fragments for amplification and immobilization for further derivatization | |
EP2240611A1 (en) | System and method for improved signal detection in nucleic acid sequencing | |
RU2810091C2 (en) | COMPOSITIONS AND METHODS OF OBTAINING LIBRARIES OF NUCLEOTIDE SEQUENCES USING CRISPR/Cas9 IMMOBILIZED ON SOLID SUPPORT | |
US20220154173A1 (en) | Compositions and Methods for Preparing Nucleic Acid Sequencing Libraries Using CRISPR/CAS9 Immobilized on a Solid Support | |
WO2023214353A1 (en) | Method of complexing biological units with particles |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20191205 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40020675 Country of ref document: HK |
|
17Q | First examination report despatched |
Effective date: 20201016 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
INTG | Intention to grant announced |
Effective date: 20230227 |
|
GRAJ | Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted |
Free format text: ORIGINAL CODE: EPIDOSDIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
INTC | Intention to grant announced (deleted) | ||
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
INTG | Intention to grant announced |
Effective date: 20230821 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE PATENT HAS BEEN GRANTED |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602018064483 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
U01 | Request for unitary effect filed |
Effective date: 20240212 |
|
U07 | Unitary effect registered |
Designated state(s): AT BE BG DE DK EE FI FR IT LT LU LV MT NL PT SE SI Effective date: 20240221 |